Novel european prrsv strain

ABSTRACT

The present invention is related to improved modified live PRRS vaccines containing new PRRSV European strains of PRRSV and methods of use and manufacture of such vaccines.

SEQUENCE LISTING

This application contains a sequence listing in accordance with 37C.F.R. 1.821-1.825. The sequence listing accompanying this applicationis hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to a live attenuated strain of a EuropeanPorcine Reproductive and Respiratory Syndrome Virus (PRRSV), methods forthe production of such strains, vaccines based thereon and methods forthe production of such vaccines and the use thereof in the treatment ofswine.

BACKGROUND OF THE INVENTION

Porcine reproductive and respiratory syndrome (PRRS) is viewed by manyas the most important disease currently affecting the pig industryworldwide. The syndrome first was described in 1987 in the United Statesas “mystery swine disease” and rapidly spread across the globe. Itcauses severe reproduction losses, is associated with increasedmortality due to secondary infections, and is linked to reduced feedconversion and average daily weight gain. Unfortunately, control of thevirus that causes PRRS has proven to be difficult.

PRRS virus (PRRSV) is an enveloped single stranded RNA virus classifiedin the family Arteriviridae (Cavanaugh, 1997). It causes a widespreaddisease of swine that was first described as ‘mystery swine disease’ inthe USA in 1987 (Hill, 1990). The disease manifests as respiratoryillness in all age groups of swine leading to death in some younger pigsand severe reproductive problems in breeding age females.

Transmission of the PRRSV can, and often does, occur through directcontact between infected and susceptible pigs. Transmission over veryshort distances by air or through semen also may occur. Once infected,the virus can remain in the blood of adults for about two weeks, and ininfected pigs for one to two months or more. Infected boars may shed thevirus in the semen for more than 100 days. This long period of viremiasignificantly increases the possibility of transmission. In addition,the PRRS virus can cross the placenta during the last third of thegestation period to infect piglets in utero and cause stillbirth orweak-born piglets.

All types and sizes of herds, including those with high or ordinaryhealth status or from either indoor or outdoor units, can be infectedwith PRRS virus. Infected herds may experience severe reproductivitylosses, as well as, increased levels of post weaning pneumonia with poorgrowth. The reproductive phase typically lasts for two to three months;however, post weaning problems often become endemic. The reproductivedisease is characterized by an abortion outbreak that affects both sowsand gilts in the last term of gestation. Premature farrowings around 109and 112 days of gestation occur. The number of stillbirths and weak-bornpiglets increases and results in a considerable increase in pre-weaningmortality.

The respiratory phase traditionally has been seen in the nursery,especially in continuous flow nurseries. However, respiratory problemscaused by PRRS virus can also be seen in the finisher as part of theporcine respiratory disease complex (PRDC). A reduction in growth rate,an increase in the percentage of unmarketable pigs, and elevated postweaning mortality can occur. Diagnostic findings indicate high levels ofpneumonia that associate with the PRRS virus together with a widevariety of other microbials commonly seen as secondary infectiousagents. Bacterial isolates may include Streptococcus suis, Haemophilussuis, Actinobacillus pleuropneumoniae, Actinobacillus suis, Mycoplasmahyopneumoniae, and Pasteurella multocida among others. Viral agentscommonly involved include swine influenza virus and porcine respiratorycorona virus. Affected pigs rarely respond to high levels of medication,and all-in/all-out systems have failed to control the disease.

PRRSV virus exists as two genotypes referred to as “US” and “EU” typewhich share about 50% sequence homology (Dea S et al. (2000). Arch Virol145:659-88). These two genotypes can also be distinguished by theirimmunological properties. Most sequencing information on variousisolates is based on the structural proteins, namely the envelopeprotein GP5 which accounts for only about 4% of the viral genome, whileonly little is known on the non-structural proteins (nsp). Isolation ofPRRSV and manufacture of vaccines have been described in a number ofpublications (WO 92/21375, WO 93/06211, WO93/03760, WO 93/07898, WO96/36356, EP 0 676 467, EP 0 732 340, EP 0 835 930).

Vaccination is the key method for alleviating the burden of PRRS as pigsthat recover from a PRRS infection will develop an immune response,which under normal circumstances will protect them from being infectedagain by the same virus strain. However, PRRS virus has the ability tochange (by mutation or recombination); and therefore, new viral strainsmay arise. In such cases, cross protection between strains may notexist, and new outbreaks may be observed in farms that had been infectedpreviously. Thus there is a continuing need for additional vaccines.

BRIEF SUMMARY OF THE INVENTION

The present invention is related to improved modified live PRRS vaccinesof European genotype and new PRRSV strains which can be used for themanufacture of such vaccines. In particular, the invention providesimproved PRRS virus strains that have been deposited with the EuropeanCollection of Cell Cultures (ECACC) under the Accession Numbers ECACC11012501 and ECACC 11012502 each deposited on Jan. 25, 2011 inaccordance with the provisions of the Budapest Treaty, or any descendantor progeny of one of the aforementioned strains.

In particular embodiments, the present invention describes a PorcineReproductive and Respiratory Syndrome Virus (PRRSV) of a European type,which is of the strain deposited with European Collection of CellCultures (ECACC) under the Accession Numbers ECACC 11012501 or AccessionNumbers ECACC 11012502.

The PRRSV is characterized in that the virus is attenuated by passagingat least 36 times in cell culture such that when the modified virus isadministered to a swine or other mammal prone to PRRSV, it fails tocause clinical signs of PRRSV disease but is capable of inducing animmune response that immunizes the mammal against pathogenic forms ofPRRSV.

Also contemplated is a method for the preparation of the live attenuatedPRRSV deposited with European Collection of Cell Cultures (ECACC) underthe Accession Numbers ECACC 11012502 or one attenuated from a parentalstrain deposited at Accession Numbers ECACC 11012501, comprisingadapting an MA 104-grown PRRSV of a European type to non-MA 104mammalian cells.

Another aspect of the invention contemplates a vaccine for theprotection of pigs against PRRSV infection, comprising the liveattenuated PRRSV deposited with European Collection of Cell Cultures(ECACC) under the Accession Numbers ECACC 11012502 or one attenuatedfrom a parental strain deposited at Accession Numbers ECACC 11012501 anda pharmaceutically acceptable carrier. Such a vaccine may advantageouslyfurther comprise one or more non-PRRSV attenuated or inactivatedpathogens or antigenic material thereof. For example, the non-PRRSVpathogens may be selected from Pseudorabies virus, Porcine influenzavirus, Porcine parvovirus, Transmissible gastroenteritis virus,Escherichia coli, Erysipelo rhusiopathiae, Bordetella bronchiseptica,Salmonella cholerasuis, Haemophilus parasuis, Pasteurella multocida,Streptococcus suis, Mycoplasma hyopneumoniae and Actinobacilluspleuropneumoniae.

In other embodiments, the vaccine may further comprise one or moreadditional European PRRSV strains selected from the group consisting ofa PRRSV strain deposited under the Accession Numbers Lelystad virusstrain (Lelystad Agent (CDI-NL-2.91), or other strains such as thosedeposited under the Accession Numbers ECACC 04102703, ECACC 04102702,ECACC 04102704, CNCM Accession No. I-1140, CNCM Accession No I-1387,CNCM Accession No I-1388, ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR2474, and VR 2402; CNCM I-1102, CNCM I-1140, CNCM I-1387, CNCM I-1388,or ECACC V93070108 or indeed may be a U.S. strain such as North AmericanPRRS virus, pT7P129A; ATCC deposit VR-2332, ATCC deposit VR-2368; ATCCVR-2495; ATCC VR 2385, ATCC VR 2386, ATCC VR 2429, ATCC VR 2474, andATCC VR 2402.

It is contemplated that the vaccine may comprise a carrier that issuitable for intradermal or intramuscular application. In someembodiments, the vaccine is in freeze-dried form. In specificembodiments, the vaccine comprises at least about 107 virus particles.

Another aspect of the invention relates to a method for the preparationof a live attenuated vaccine for combating PRRS, comprising admixing alive attenuated PRRSV virus deposited with European Collection of CellCultures (ECACC) under the Accession Number ECACC 11012502 or oneattenuated from a parental strain deposited at Accession Numbers ECACC11012501 with a pharmaceutically acceptable carrier. In such methods thelive attenuated PRRSV may preferably further comprise one or moreadditional European PRRSV strains selected from the group consisting ofa PRRSV strain deposited under the Accession Numbers ECACC 04102703,ECACC 04102702, ECACC 04102704, CNCM Accession No. I-1140, CNCMAccession No I-1387, and CNCM Accession No I-1388.

In some embodiments, the live attenuated PRRSV may further comprise anadjuvant.

Also contemplated is a method of immunizing swine against porcinereproductive and respiratory syndrome (PRRS), the method comprising thestep of administering to swine a vaccine composition including a liveporcine reproductive and respiratory syndrome virus mixed with apharmacologically compatible carrier agent, the virus comprising PRRS94881 virus passaged at least 36 times in cell culture to modify thevirus such that when the modified virus is administered to a swine orother mammal prone to PRRS, it fails to cause clinical signs of PRRSdisease but is capable of inducing an immune response that immunizes themammal against pathogenic forms of PRRS.

In some embodiments, the method is performed wherein the swine presentsno lung lesions after vaccination. In other embodiments, the swinepresents fewer lung lesions after vaccination as compared to vaccinationwith Porcilis vaccine.

Another aspect of the invention relates to a PRRS virus having anucleotide sequence that is at least 95% homologous with the sequenceset forth in either SEQ ID NO:1 or SEQ ID NO:10.

Also contemplated is a PRRS virus that comprises at least one ORF thatencodes a protein that is at least 98% identical to any of the sequencesset forth in SEQ ID NO: 2 to 9 or SEQ ID NO:11 to SEQ ID NO:18.

Also contemplated is a PRRS virus that has a nucleotide sequence of SEQID NO:1 or SEQ ID NO:10 or a fragment of either SEQ ID NO:1 or SEQ IDNO:2 wherein the fragment encodes an ORF selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17,and SEQ ID NO:18.

The invention further relates to a subunit vaccine for vaccination of aporcine animal wherein the vaccine comprises one or more nucleotidesselected from the group consisting of a nucleotide that encodes an ORFselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.

Another aspect of the invention relates to a subunit vaccine forvaccination of a porcine animal wherein the vaccine comprises one ormore nucleotides selected from the group consisting of SEQ ID NO:19; SEQID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ IDNO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ IDNO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; and SEQ ID NO:34.

Also contemplated is a composition comprising one or more proteinsselected from the group consisting of a protein having the sequence ofSEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ IDNO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQID NO:18.

Also contemplated is an isolated nucleic acid comprising a sequenceselected from the group consisting of SEQ ID NO:19; SEQ ID NO:20; SEQ IDNO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ ID NO:25; SEQ IDNO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ ID NO:30; SEQ IDNO:31; SEQ ID NO:32; SEQ ID NO:33; SEQ ID NO:34.

The invention further relates to a recombinant expression vector and/orvaccine comprising such expression vectors, wherein said vectorscomprise a nucleic acid sequence that encodes one or more PRRSV ORFsselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18 operably linked to apromoter. In such embodiments, the nucleic acid encoding the ORFs maypreferably be selected from the group consisting of SEQ ID NO:19; SEQ IDNO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ IDNO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ IDNO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; and SEQ ID NO:34.

BRIEF DESCRIPTION OF SEVERAL VIEWS OF THE DRAWINGS

FIG. 1A: Clinical observation of cough score in respiratory challengemodel using European challenge strain.

FIG. 1B: Clinical observation of total clinical score in respiratorychallenge model using a European challenge strain.

FIG. 2: Rectal temperature measurements in respiratory challenge modelusing a European challenge strain.

FIG. 3: Average daily weight gain measurements in respiratory challengemodel using a European challenge strain.

FIG. 4: PRRS viremia as indicated through quantitative PCR inrespiratory challenge model using a European challenge strain.

FIG. 5: PRSS Serology as indicated by ELISA in respiratory challengemodel using a European challenge strain.

FIG. 6: Macroscopic examination of lung lesions in respiratory challengemodel using a European challenge strain.

FIG. 7A-C: Histopathology measurements. FIG. 7A shows means macroscopiclung lesions; FIG. 7B shows control animal histopathology; FIG. 7B showsPRRS infected animal histopathology.

FIG. 8: Shows RT-PCT Time PCR results depicting % viremia in animalsvaccinated with EU PRRS 94881.

FIG. 9: Concurrent process for large-scale production of EU PRRS 94881.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods of treating or reducing theseverity of porcine reproductive and respiratory syndrome virus (PRRSV)infection, as well as, methods of preventing PRRSV infection. Generally,the method is for treating or reducing the severity of or incidence ofporcine reproductive and respiratory syndrome virus (PRRSV) infection.“Treating or reducing the severity of or incidence of” refers to areduction in the severity of clinical signs, symptoms, and/orpathological signs normally associated with infection, up to andincluding prevention of any such signs or symptoms. “Pathological signs”refers to evidence of infection that is found microscopically or duringnecropsy (e.g. lung lesions).

The method generally includes the step of administering a therapeuticamount of a PRRSV antigen to a swine of a defined age or age range. Forexample, in one aspect of the invention, one therapeutic amount of aPRRSV antigen may be administered to a piglet about three-weeks-old oryounger, and different therapeutic amounts of the antigen may beadministered to a pig between about 3 weeks of age and 4 weeks of age.Similarly, an even different therapeutic amount might be administered toa pig between about four weeks and sixteen weeks of age (or any agewithin this range, e.g. five weeks to six weeks of age, nine weeks tofifteen weeks of age, seven weeks to ten weeks of age, etc.), or to pigolder than sixteen weeks, such as an adult sow.

In specific embodiments, the present invention relates to an attenuated,atypical PRRSV strain and corresponding improved modified-live vaccineswhich confer effective immunity to this newly discovered typical PRRSVstrain. “Effective immunity” refers to the ability of a vaccine toprevent swine PRRSV infections, including atypical PRRSV infections,which result in substantial clinical signs of the disease. It should beunderstood that the immunized swine may or may not be serologicallypositive for PRRSV, but the swine do not exhibit any substantialclinical symptoms.

In preferred forms, the vaccine of the invention includes a liveEuropean type PRRS live virus which has been attenuated in virulence.The resulting attenuated virus has been shown to be avirulent inchallenged controlled host animal studies and to confer effectiveimmunity. This particular strain of EU PRRS is not as virulent as othersand hence, it is an attractive option as a vaccine candidate. The PRRSV94881 parental strain does not cause severe, atypical PRRS disease inthe pregnant sow nor severe lung lesions in young pigs. This strain wasinitially isolated from in the North Rhine Westphalia, Germany from a 3week old piglet with severe respiratory disorder. The strain wassubsequently attenuated via continuous passage in MA 104 cells. Theattenuated strain was deposited in the European Collection of CellCultures (ECACC), Porton Down, Salisbury, Wiltshire, SP4 0JG, GreatBritain, on Jan. 25, 2011 and was accorded Accession No. 11012502. Thisattenuated virus is a preferred Master Seed Virus (MSV) which has beensubsequently passaged and developed as an effective PRRSV vaccine. Thevirulent parent strain denominated 94881 also was deposited inaccordance with the Budapest Treaty at the European Collection of CellCultures (ECACC), Porton Down, Salisbury, Wiltshire, SP4 0JG, GreatBritain, on Jan. 25, 2011 and was accorded Accession No. 11012501.

In certain exemplary embodiments the modified live virus vaccine wastested at a dosage of 1 ml for pigs and 2 ml for sows via intramuscularinjection and was shown to be efficacious in producing protectiveimmunity.

Passaging of the virus to attenuation was accomplished using classicalvirology methods. Specifically, the parental isolate PRRS 94881 wasattenuated in vitro through continuous passing in MA 104 cells toachieve a maximum passage of 108 passes past initial isolation. Briefly,the material was passed at roughly 1 to 2 passes per week for a total of108 passes in T-25 cm² or T-75 cm² flasks. Confluent MA 104 cellcultures with approximately 12-30 mL of Minimal Essential Medium (MEM)supplemented with 6% fetal bovine serum (FBS) were inoculated with 100to 300 μl of the virus. Cultures were incubated for 3-7 days in ahumidified chamber incubator at 37° C. with 4-6% CO₂. Once culturesreached >25% cytopathic effect (CPE), the flask was harvested byextracting the supernatant. A portion of the supernatant was passed intoa new flask and 2 mL of the harvest was aliquoted for storage at −60° C.to −80° C.

The skilled person using standard techniques in the art will be able todetermine the underlying nucleic acid sequence of the attenuated virushas been deposited ECACC under Accession No. 11012502. The presentinvention therefore further embraces a nucleic acid sequence specificfor the attenuated PRRSV 94481 deposited at ECACC under Accession No.11012502. Preferably, the invention further embraces PRRS virus nucleicacid sequences that share at least 95% sequence homology with thesequence of SEQ ID NO:1 or SEQ ID NO:10 as such viruses may likely beeffective at conferring immunity upon animals vaccinated with attenuatedviruses containing such homologous sequences. The sequence shown in SEQID NO:1 is the full length sequence of the attenuated PRRS 94881 MSV andhas a full length sequence of 14843 bp. The ORFS1 through 7 have beenannotated for this sequence as follows:

ORF Number CDS in SEQ ID NO: 1 Protein encoded ORF1a  178 to 7227 SEQ IDNO: 2 ORF1b  7209 to 11600 SEQ ID NO: 3 ORF2 11611 to 12360 SEQ ID NO: 4ORF3 12219 to 13016 SEQ ID NO: 5 ORF4 12761 to 13312 SEQ ID NO: 6 ORF513309 to 13914 SEQ ID NO: 7 ORF6 13902 to 14423 SEQ ID NO: 8 ORF7 14413to 14799 SEQ ID NO: 9

The sequence shown in SEQ ID NO:10 is the full length sequence of theparental PRRSV 94881 strain, passage 5 and has a full length sequence of14843 bp. The ORFS1 through 7 have been annotated for this sequence asfollows:

ORF Number CDS in SEQ ID NO: 10 Protein encoded ORF1a  178 to 7227 SEQID NO: 11 ORF1b  7209 to 11600 SEQ ID NO: 12 ORF2 11611 to 12360 SEQ IDNO: 13 ORF3 12219 to 13016 SEQ ID NO: 14 ORF4 12761 to 13312 SEQ ID NO:15 ORF5 13309 to 13914 SEQ ID NO: 16 ORF6 13902 to 14423 SEQ ID NO: 17ORF7 14413 to 14799 SEQ ID NO: 18

With the isolation of this new attenuated European PRRS virus strain itis possible to produce improved PRRS vaccines containing a most recentPRRS strain that is reflective of virulent PRRS strains found currentlyin the field. In particular, the new attenuated European PRRS virus maybe used to prepare modified live vaccines (MLV). A modified live vaccineis characterized in that it contains live virus which can replicate inpigs, but does not exert clinical disease of PRRS. Furthermore, uponadministration it induces an immunological response in pigs whichgenerally leads to a significant extent of protection against subsequentinfection with pathogenic PRRS virus. Virus showing such characteristicsis usually called attenuated virus. In addition, the present inventionprovides details of the sequences of the ORFs of both the parental andthe attenuated strains of PRRSV 94881. Thus, it is contemplated that theskilled person may employ the sequences of any one or more of the ORFsshown herein in a subunit vaccine.

As noted above, in general, attenuation of virus may be generated frompathogenic virus isolates by repeated passaging in suitable host cellsthat are permissive to the virus until the virus shows the desiredproperties (WO 92/21375, WO 93/06211, WO93/03760, WO 93/07898, WO96/36356, EP 0 676 467, EP 0 732 340, EP 0 835 930). Alternatively, itmay be generated by genetic reengineering through use of an infectiousclone, normally a full-length complementary DNA transcript of the viralgenome (WO 98/18933, EP 1 018 557, WO 03/062407, Nielsen et al, J Virol2003, 77:3702-371 1). In a preferred embodiment, the present inventionrelates to a MLV containing attenuated PRRS virus of European genotype94481 that is attenuated from a parental virus that is deposited atECACC under Accession No. 11012501. A preferred MLV contains theattenuated virus of the present invention that is deposited at ECACCunder Accession No. 11012502.

In another aspect, the present invention contemplates preparation andisolation of a progeny or descendant of a PPRS virus that has beendeposited on Jan. 25, 2011 with the European Collection of Cell Cultures(ECACC), Porton Down, Salisbury, Wiltshire, SP4 0JG, Great Britain,under the Accession Numbers ECACC 11012502 (attenuated strain for MLV)and 11012501 (parental strain). The invention therefore extends to PRRSvirus strains which are derived from the deposited strains throughpropagation or multiplication in an identical or divergent form, inparticular descendants which possess the essential characteristics ofthe deposited strains. Upon continued propagation, the strains mayacquire mutations most of which will not alter the properties of thesestrains significantly.

The strains of the invention may also be further modified to impartfurther desirable properties to them. This may be achieved by classicalpropagation and selection techniques, like continued propagation insuitable host cells to extend the attenuated phenotype. Alternatively,the strains may be genetically modified by directed mutation of thenucleic acid sequence of the genome of these strains by suitable geneticengineering techniques. The genome of PRRSV was completely or partlysequenced (Conzelmann et al., 1993; Meulenberg et al., 1993a, Murtaughet al, 1995) and encodes, besides the RNA dependent RNA polymerase (ORFs1a and 1b), six structural proteins of which four envelope glycoproteinsnamed GP2 (ORF2), GP3 (ORF3), GP4 (ORF4) and GP5 (ORF5), anon-glycosylated membrane protein M (ORF6) and the nucleocapsid proteinN(ORF7) (Meulenberg et al. 1995, 1996; van Nieuwstadt et al., 1996).Immunological characterization and nucleotide sequencing of European andUS strains of PRRSV has identified minor antigenic differences withinstrains of PRRSV located in the structural viral proteins (Nelson etal., 1993; Wensvoort et al., 1992; Murtaugh et al., 1995). The PRRS94881 MSV of the present invention has been compared with the EuropeanReference Virus strain Lelystad Virus (LV) revealed nucleotidehomologies ranging from 85.40 to 95.09 percent in the 8 different viralgenes and amino acid identities from 86.39 to 97.27 percent between bothvirus strains. Two deletions in the ORF 1a of 94881 MSV could beidentified compared to LV. For example, ORF1a of 94881 MSV has 85.40%nucleotide homology to Lelystad Virus resulting in an amino acididentity of 86.39%; ORF1b of 94881 MSV has 92.12% nucleotide homology toLelystad Virus resulting in an amino acid identity of 97.27%; ORF2 of94881 MSV has 91.07% nucleotide homology to Lelystad Virus resulting inan amino acid identity of 90.76%; ORF3 of 94881 MSV has 90.98%nucleotide homology to Lelystad Virus resulting in an amino acididentity of 89.43%; ORF4 of 94881 MSV has 90.58% nucleotide homology toLelystad Virus resulting in an amino acid identity of 87.43%; ORF5 of94881 MSV has 90.43% nucleotide homology to Lelystad Virus resulting inan amino acid identity of 88.56%; ORF6 of 94881 MSV has 95.02%nucleotide homology to Lelystad Virus resulting in an amino acididentity of 97.11%; ORF7 of 94881 MSV has 95.09% nucleotide homology toLelystad Virus resulting in an amino acid identity of 92.97%;

Indeed, the PRRS 94881 virus of the present invention may be made into achimeric virus wherein the backbone of the PRRS virus under ECACCAccession No. 11012502 or indeed the parent strain deposited under ECACCAccession No 11012501 is modified to replace the endogenous sequence ofone or more of ORF 1a, ORF 1b, ORF 2, ORF 3, ORF 4, ORF 5, ORF 6, or ORF7 with the corresponding ORF from a different strain of PRRS virus. Forexample, the different strain of the PRRS virus may be a differentEuropean strain such as Lelystad virus strain (Lelystad Agent(CDI-NL-2.91), or other strains such as those deposited under theAccession Numbers ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCMAccession No. I-1140, CNCM Accession No I-1387, CNCM Accession NoI-1388, ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR 2474, and VR 2402;CNCM I-1102, CNCM I-1140, CNCM I-1387, CNCM I-1388, or ECACC V93070108or indeed may be a U.S. strain such as North American PRRS virus,pT7P129A; ATCC deposit VR-2332, ATCC deposit VR-2368; ATCC VR-2495; ATCCVR 2385, ATCC VR 2386, ATCC VR 2429, ATCC VR 2474, and ATCC VR 2402.

Recombinant techniques for preparing modified sequences are well knownto those of skill in the art and usually employ construction of afull-length complementary DNA copies (infectious clones) of the viralgenome which may then be modified by DNA recombination and manipulationmethods (like site-directed mutagenesis etc.). This way, for exampleantigenic sites or enzymatic properties of viral proteins may bemodified. Infectious clones of PRRS virus strains of European and NorthAmerican genotype have been reported in the literature.

The PRRS virus strains of the present invention are suitable forvaccines of the invention can be grown and harvested by methods known inthe art, e.g. by propagating in suitable host cells like the simian cellline MA-104, Vero cells, or porcine alveolar macrophages. PRRSVpreferentially grows in alveolar lung macrophages (Wensvoort et al.,1991). A few cell lines, such as CL2621 and other cell lines cloned fromthe monkey kidney cell line MA-104 (Benfield et al., 1992; Collins etal., 1992; Kim et al., 1993) are also susceptible to the virus.

Vaccines comprising any one of PRRSV strain PRRS virus under ECACCAccession No. 11012501, 11012501 Accession Numbers ECACC 04102703, ECACC04102702, ECACC 04102704, CNCM Accession No. I-1140, CNCM Accession NoI-1387, and CNCM Accession No I-1388 as well as any combination of thesestrains or their descendants are thus preferred embodiments of thepresent invention. In specific embodiments, the PRRS virus 94881 isgrown in a process wherein there is a concurrent seeding of the virusand the host cells together on the same day into a bioreactor as shownin FIG. 9. Additional features of a method of production of PRRS virus94881 may be as described in concurrently filed herewith as U.S.Provisional entitled “A Commercial scale process for production ofPRRSV” filed concurrently with the instant application under applicationNo. 61/444,071. While this is one method of growing the PRRSV 94881, itshould be understood that the virus may be propagated according to anyconventional method known to those of skill in the art.

Preferably, vaccines according to the present invention are modifiedlive vaccines comprising one or more of these strains alive in asuitable carrier, but inactivated virus may also be used to preparekilled vaccine (KV). MLV are typically formulated to allowadministration of 10¹ to 10⁷ viral particles per dose, preferably 10³ to10⁵ particles per dose, more preferably 10⁴ to 10⁵ particles per dose(4.0-5.0 log₁₀ TCID₅₀). KV may be formulated based on a pre-inactivationtiter of 10³ to 10¹⁰ viral particles per dose. The vaccine may comprisea pharmaceutically acceptable carrier, for example a physiologicalsalt-solution.

Pigs can be infected by PRRSV via the oronasal route. Virus in the lungsis taken up by lung alveolar macrophages and in these cells replicationof PRRSV is completed within 9 hours. PRRSV travels from the lungs tothe lung lymph nodes within 12 hours and to peripheral lymph nodes, bonemarrow and spleen within 3 days. At these sites, only a few cells stainpositive for viral antigen. The virus is present in the blood during atleast 21 days and often much longer. After 7 days, antibodies to PRRSVare found in the blood. The combined presence of virus and antibody inPRRS infected pigs shows that the virus infection can persist for a longtime, albeit at a low level, despite the presence of antibody. During atleast 7 weeks, the population of alveolar cells in the lungs isdifferent from normal SPF lungs.

A vaccine according to the present invention may be presented in form ofa freeze-dried preparation of the live virus, to be reconstituted with asolvent, to result in a solution for injection. The solvent may e.g. bewater, physiological saline, or buffer, or an adjuvanting solvent. Thesolvent may contain adjuvants. The reconstituted vaccine may then beinjected into the a pig, for example as an intramuscular or intradermalinjection into the neck. For intramuscular injection, a volume of 2 mlmay be applied, for an intradermal injection it is typically 0.2 ml. Ina further aspect, the present invention therefore is a vaccine product,comprising in separate containers a freeze-dried composition of thevirus, and a solvent for reconstitution, and optionally furthercontaining a leaflet or label comprising instructions of use.

A vaccine according to the present invention may not only comprise oneor more of the aforementioned strains, but may include furthercomponents active against PRRS or other porcine viral or bacterialdiseases, like porcine circovirus or classical swine fever virus.Therefore, the invention further relates to a vaccine as described,characterized in that it contains at least one further antigen activeagainst a porcine disease which is not PRRS. For example, such furtherantigens may include Mycoplasma hyopneumoniae, PCV2, SIV, H. parasuis,E. rhusiopathiae, S. suis, A. suis, Leptospira sp. Parvovirus and thelike. In addition, the vaccine may comprise certain pharmaceutically orveterinary acceptable adjuvants. The invention provides new vaccinecompositions, in particular, PRRS virus vaccines comprising PRRSV 94881that further comprise adjuvants that enhance the efficacy of the vaccinesuch that a better clinical response/outcome is seen with theadministration of the combination of the adjuvant and the vaccine ascompared to administration of the vaccine alone. For example, thevaccine compositions of the invention may comprise a PRRSV 94881 virusvaccine and an adjuvant selected from the group consisting of MCP-1,α-tocopherol (e.g., α-tocopherol acetate, an exemplary version of whichis sold as DILUVAC FORTE®), Haemophilus sonmus fractions, carbopol andcombinations thereof. In some embodiments, the virus vaccine comprisingPRRS 94881 virus vaccine, which may be a recombinant subunit vaccine oralternatively may be a live attenuated virus vaccine. An exemplary livevaccine that exists is INGELVAC® PRRS MLV and the PRRS 94881 may beformulated in a manner similar to INGELVAC® PRRS MLV.

In addition to the above, the immunogenic compositions of the inventionmay contain other ingredients so long as the other ingredients do notinterfere with the adjuvants or the underlying virus vaccine. Such otheringredients include, for example, binders, colorants, desiccants,antiseptics, wetting agents, stabilizers, excipients, adhesives,plasticizers, tackifiers, thickeners, patch materials, ointment bases,keratin removers, basic substances, absorption promoters, fatty acids,fatty acid ester, higher alcohols, surfactants, water, and bufferagents. Preferred other ingredients include buffer agents, ointmentbases, fatty acids, antiseptics, basic substances, or surfactants.

The content or amount of the adjuvants used in the invention may varyand can be determined by taking into consideration, for example, theproperties of the PRRS virus vaccine being used, and the dosage form.The adjuvant may comprise, for example, 1 to 100% by weight. The PRRSV94881-based compositions of the invention are produced by mixingtogether the adjuvant component and the virus vaccine component, eitheralone or with various other ingredients. The compositions may be suchthat the virus vaccine and the adjuvant are presented as one formulationor alternatively, the adjuvant and the vaccine are presented in distinctformulations that can be administered simultaneously or sequentially.

The adjuvant component of the immunogenic compositions of the inventionthus may be administered separately from the virus vaccine in theadministration to organisms. Alternatively, the adjuvant according tothe present invention, together with the virus vaccine, can beadministered as a single vaccine composition. The virus vaccine may beany virus vaccine. More specific embodiments contemplate the use of aPRRS virus vaccine comprising PRRSV 94881. In addition such a vaccinemay be combined with other vaccines such as INGELVAC® PRRS MLV and/orPorcilis®. This is merely one exemplary PRRS virus combination vaccineand other such vaccine combinations can be readily prepared.

The immunogenic compositions described herein are particularlyadvantageous in the induction of the production of an antibody responseto PRRS virus. Administration of the vaccines preferably will produce alessening of the severity of one or more clinical symptoms, such as lunglesions, anorexia, skin discolorations, lethargy, respiratory signs,mummified piglets, coughing, diarrhea and combinations thereof, that areassociated with PRRSV infection.

The compositions thus particularly enhance the clinical outcome in adiseased animal as compared to the outcome from administration of PRRSvirus vaccine alone. In specific embodiments, the enhanced clinicaloutcome is a reduction of the percentage of lung lesions by at least 50%when compared to animals not receiving the immunogenic composition incombination with said adjuvant. In other embodiments, the enhanceclinical outcome is a reduction of viremia in animals by at least 45%when compared to animals not receiving the immunogenic composition incombination with said adjuvant.

Thus, in one aspect, the invention relates to an improved vaccine, moreparticularly and improved PRRS virus vaccine, wherein the improvementcomprises admixing with the virus vaccine an adjuvant selected from thegroup consisting of MCP-1, Haemophilus sonmus fractions, carbopol andcombinations thereof. The vaccine composition of the invention mayfurther comprise a pharmaceutically acceptable carrier.

The vaccine compositions of the invention may be formulated by anymethod known in the art of formulation, for example, into liquidpreparations, suspensions, ointments, powders, lotions, W/O emulsions,0/W emulsions, emulsions, creams, cataplasms, patches, and gels and ispreferably used as medicaments. Thus, according to another aspect of thepresent invention, there is provided a pharmaceutical compositioncomprising the above vaccine composition. The vaccine compositionaccording to the present invention, when dermally administered, cansignificantly induce antibody production. Accordingly, in anotherpreferred embodiment of the present invention, the vaccine compositioncan be provided as a transdermal preparation.

Further, as described above, the virus and adjuvant in the presentinvention may be administered, to an organism, together as a singlevaccine composition, or as an adjuvant preparation separate and distinctfrom the antigenic PRRS virus component of the vaccine, whereby theadjuvant acts in a manner such that amount of an antibody produced inthe organism in response to the PRRS virus vaccine can be significantlyincreased as compared to administration of the PRRS virus vaccine alone.

When the adjuvant and the PRRS virus vaccine are administered to anorganism, the clinical outcome of the animal is enhanced. The effectiveamount of the adjuvant and the immunologically effective amount of thePRRS virus vaccine may be readily determined by a person having ordinaryskill in the art by taking into consideration, for example, the type andproperties of the antigenic substance, the species of organisms, age,body weight, severity of diseases, the type of diseases, the time ofadministration, and administration method and further using the amountof an antibody produced against the antigenic substance in the organismas an index.

The PRRS virus vaccine, the adjuvant, or combinations thereof can beadministered to organisms by any suitable method selected depending, forexample, upon the condition of animals and properties of diseases.Examples of such methods include intraperitoneal administration, dermaladministration for example, subcutaneous injection, intramuscularinjection, intradermal injection, and patching, nasal administration,oral administration, mucosal administration (for example, rectaladministration, vaginal administration, and corneal administration).Among them, intramuscular administration is preferred.

An exemplary therapeutic dose of PRRSV MLV is about two milliliters (2mLs). Skilled artisans will recognize that the dosage amount may bevaried based on the breed, size, and other physical factors of theindividual subject, as well as, the specific formulation of PRRSV MLVand the route of administration. Preferably, the PRRSV MLV isadministered in a single dose; however, additional doses may be useful.Again, the skilled artisan will recognize through the present inventionthat the dosage and number of doses is influenced by the age andphysical condition of the subject pig, as well as, other considerationscommon to the industry and the specific conditions under which the PRRSVMLV is administered.

In certain other embodiments, the vaccine may be a multivalent vaccinethat comprises two or more PRRS viruses where at least one of the PRRSviruses is the attenuated 94881 virus deposited under ECACC AccessionNo. 11012502. The other PRRS viruses may be one or more selected fromthe group consisting of PRRSV strain Lelystad virus (Lelystad Agent(CDI-NL-2.91), or other strains such as those deposited under theAccession Numbers ECACC 04102703, ECACC 04102702, ECACC 04102704, CNCMAccession No. I-1140, CNCM Accession No I-1387, CNCM Accession NoI-1388, ATCC VR 2332, VR 2385, VR 2386, VR 2429, VR 2474, and VR 2402;CNCM I-1102, CNCM I-1140, CNCM I-1387, CNCM I-1388, or ECACC V93070108or indeed may be a U.S. strain such as North American PRRS virus,pT7P129A; ATCC deposit VR-2332, ATCC deposit VR-2368; ATCC VR-2495; ATCCVR 2385, ATCC VR 2386, ATCC VR 2429, ATCC VR 2474, and ATCC VR 2402.

The vaccines based on PRRS viruses may be used to vaccinate both pigletsand sows. In one aspect of the invention, a particular dose regimen isselected based on the age of the pig and antigen selected foradministration. This will permit pigs of any age to receive the mostefficacious dose. In a preferred method, a therapeutic amount of PRRSV94881 MLV is administered to a pig or piglet that is about two weeksold±5 days of age. The amount selected will vary depending upon the ageof the pig. Alternatively, a different therapeutic amount of such an MLVis administered to a pig or piglet that is older than about 3 weeks, andthis amount will also change as the pig receiving such an administrationages or becomes older. Accordingly, pigs about four weeks old, six weeksold, eight weeks old, ten weeks old, twelve weeks old, fourteen weeksold, sixteen weeks old, a gilt, or a sow will all receive differentamounts. Therapeutic dose to be used will be optimized in the field andis typically, determined in clinical studies in which a minimalimmunizing dose is defined based on protection against a virulentheterologous PRRSV challenge in susceptible swine. Preferably, the PRRSVMLV produced according to the methods described herein is administeredintramuscular administration; however, other methods of administrationsuch as intradermal, intranasal, intraretinal, oral, subcutaneous, andthe like, that are well-known and used in the art may be used.

The skilled person will recognize that the vaccination methods mayinvolve determining the proper timing and dosage for vaccination of apig against PRRSV. Such methods generally comprises the steps ofdetermining at least one variable selected from the group consisting ofage, health status, innate immunity level and active immunity level, ofthe pig, and adjusting a standard dosage level to account for thesevariables. Generally, the innate immunity level and active immunitylevel will be determined by referring to a standard comprised of averagelevels from a population of pigs of similar age and health status. In aparticularly preferred method, all variable are considered prior todetermining the optimum dosage level and timing of administration.

In preferred embodiments, the present invention also relates to isolatednucleic acids that code specific open reading frames of attenuated 94881virus deposited under ECACC Accession No. 11012502 and the parentvirulent 94881 virus deposited under ECACC Accession No. 11012501. Forexample, the complete nucleotide sequence of the attenuated 94881 virusdeposited under ECACC Accession No. 11012502 has a sequence of SEQ IDNO:1 which encodes ORF1a, ORF1b, ORF2, ORF3, ORF4, ORF5, ORF6, ORF7protein sequences of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5,SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.The complete nucleotide sequence of the parent virulent 94881 virusdeposited under ECACC Accession No. 11012501 has a sequence of SEQ IDNO:10 which encodes ORF1a, ORF1b, ORF2, ORF3, ORF4, ORF5, ORF6, ORF7protein sequences of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ IDNO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18,respectively.

The PRRSV 94881 vaccine can be administered in any conventional fashionand in some preferred methods the administration is intramuscularly. Itis preferred that the administered PRRSV vaccine provide its benefits oftreating or reducing the severity of or incidence of PRRSV infectionafter a single dose, as with INGELVAC®, however, if other antigens orcombination or multivalent vaccines are selected, it should beunderstood that they can be administered in their conventional fashion,which may include one or more booster doses after the initialadministration. Those of skill in the art will be able to determineappropriate dosing levels based on the PRRSV vaccine selected and theage range of the animal to which the antigen will be administered.

In specific examples presented herein below pigs and sows werechallenged with a new European derived strain of PRRSV that was able toreproducibly produce respiratory disease in piglets. Historically,European-derived PRRSV strains have been unable to reproduce respiratorydisease in piglet model and hence respiratory challenge models relied oninfection with non-European strains. Because of high genetic diversitythere is a demand in Europe for a new vaccine based on an Europeanstrain. In additional examples, the animals were challenged with astrain that produced reproductive failure in gilts/sows challengemodels. It has been found that the efficacy of the MLV vaccines based onattenuated 94881 virus deposited under ECACC Accession No 11012502 orany virus prepared from this strain or from the parental straindeposited at ECACC Accession No 11012501 can be shown using a variety ofchallenge models because this strain also is effective in other modelsof PRRS virus-induced respiratory or reproductive failure.

EXAMPLES Example 1 Description of PRRSV Respiratory Challenge Model

As noted above, historically, EU-derived PRRSV strains are unable toreproduce respiratory disease in piglet model. Because of the highgenetic diversity there is a demand in Europe for a new vaccine based ona European strain and a good, reproducible respiratory challenge modelutilizing virulent a European-derived PRRSV strain is necessary for theconduction of the studies. In the following example, the inventors showthat challenge of pigs with low passage European challenge strain(passage 4) reliably produced respiratory symptoms.

In this study, 3 groups with 12 animals, 3 weeks of age at allocationand appr. 10 weeks of age at challenge were used:

Group 1: Control group

Group 2: Challenge group (SD 35)

Group 3: Vaccinated with PORCILIS®PRRSV (SD 0) and then challenged (SD35).

The study was conducted over a 56 day period necropsy of 6 animals fromeach group was measured 10 days after challenge; necropsy of allremaining animals 21 days after challenge. Daily investigated parametersincluded: rectal temperature, respiratory and other clinical signs.Further parameters investigated included: body weight, mortality,viremia, seroconversion, pathological and histological examination ofthe lungs.

At study day −7 the groups of pigs were allocated to each group. Atstudy day 0, Group 3 was vaccinated with PORCILIS®PRRSV. At study day 35group 2 and group 3 were challenged with a European challenge strain. Atday 45 6 animals from each group were euthanized. The remaining animalswere euthanized at day 56.

FIG. 1A shows coughing measurements as a meanscore per animal and week.It was seen that there was an increase in coughing after challenge inboth the challenge alone (Group 2) and the Porcilis group (Group 3).FIG. 1B shows the total clinical score which was taken from dyspnea,coughing, nasal and eye discharge and behavior. These data showed thatthere was an overall increase in total clinical score after challenge inthe challenge and Porcilis group. Rectal temperature of the animals wasmonitored before and after challenge and shows that there was anincrease rectal temperature in the challenge and Porcilis group afterChallenge (SD>35 group 1-2 p≦0.001; group 1-3 p≦0.001) (FIG. 2).

Measurement of average daily weight (FIG. 3) showed that after challengeuntil first and until second necropsy the ADW was significantly less inthe challenge (SD35-44 p≦0.001 and SD35-56 p≦0.01) and in the Porcilisvaccinated group (SD35-44 p≦0.05 and SD35-56 p≦0.05).

Viremia was monitored using PCR (FIG. 4) and ELISA assays (FIG. 5). PCRshowed that in Group 1: all animals from the control group remainednegative. In Group 2: all animals were positive for PRRSV afterchallenge; in Group 3: all animals were positive for PRRSV aftervaccination. ELISA revealed: that in Group 1: all animals remainednegative; in Group 2: all animals were positive for PRRSV AB afterchallenge in Group 3: all animals were positive for PRRSV AB aftervaccination.

Macroscopic examination of the lungs was also performed (FIG. 6) wherelungs were evaluated for tan mottled areas and areas of consolidation:In comparison to the control group significant macroscopic changes couldbe observed in the challenge and Porcilis group (group 1-2 p≦0.001;group 1-3 p≦0.05). In histopathological examination, the data alsoshowed efficacy of vaccination (FIG. 7A through 7C). Mean lung lesionscore was significantly higher in the challenge and Porcilis group incomparison to the control group (group 1-2 p≦0.001; group 1-3 p≦0.001).Microscopic lesions were stronger 10 days post infection.

In summary, coughing, total clinical scores and rectal temperaturesincreased after challenge in the challenge controls and Porcilisvaccinated groups. Body weight was significantly (p<0.05) less in thechallenge controls and Porcilis group in comparison to the negativecontrol group. All animals from the Porcilis group were positive forPRRS virus and antibodies after vaccination. All animals from thechallenge controls were positive for PRRS virus and antibodies afterchallenge. Macroscopical and histological analysis of the lungs showedsevere macroscopic and microscopic lung lesions in the challengecontrols and Porcilis group in comparison to the negative control group.

This study thus confirmed that the European challenge strain used doesinduce significant (p<0.05) disease when compared to the negativecontrol group: fever, coughing, reduced weight and severe macroscopicand microscopic lung lesions.

In addition, the European challenge strain has successfully demonstratedconsistent and reproducible PRRSV-specific respiratory disease in thepig challenge model and therefore, is suitable for use as a challengevirus in future efficacy studies. Porcilis PRRS shows a lack ofefficiency against the European challenge strain within the parametersof this study.

Example 2 Evaluation of Minimum Immunizing Dose of Attenuated PRRS Virus94881 in Susceptible 2 Week Old Piglets Following Challenge withHeterologous European PRRS Isolate

A vaccination-challenge study was performed to evaluate the minimumimmunizing dose (MID) of Porcine Reproductive and Respiratory SyndromeVaccine European-derived isolate 94881, Modified Live Virus (PRRS 94881MLV) at three different titer levels, administered to PorcineReproductive and Respiratory Syndrome (PRRS) susceptible piglets, thatwere approximately 14 days of age, to provide relevant reduction in lunglesions following challenge with a heterologous European isolate ofPRRS. Fifteen piglets were included in each vaccinated group (Groups1-3) as well as in the challenge control group (Group 4). Ten pigletswere included in the negative control group (Group 5).

The vaccine groups and the challenge control groups were monitored for anumber of parameters including: viremia post-challenge, clinicalassessments post-vaccination, PRRS serology, viremia post-vaccination,clinical observations post-challenge, average daily weight gain (ADWG),rectal temperatures and PRRS virus detection in the lungs. A negativecontrol group (Group 5), which was not challenged, was also included inthe study for study validation purposes by demonstrating thatbiosecurity was not breached throughout the duration of the study.

The challenge control and negative control groups were PRRS negative upto the day of challenge (D28) and the negative control group remainedPRRS negative for the remainder of the study (D38), thus validating thestudy.

Challenge was at 4 weeks post-vaccination. At this time, only 2 animalsin low titer vaccine group, 1 animal in the medium titer vaccine groupand 3 animals in the high titer vaccine group were PRRS qPCR positive inserum.

The challenge control group exhibited significant lung lesions typicalof PRRS post-challenge. Post-challenge, the low, medium and high titervaccine groups had median total lung lesion scores of 0.13%, 0.55%, and0.40%, respectively; while the challenge control group had a mediantotal lung lesion score of 33.40%. The median total lung lesion scoresfor the three vaccine titer groups were significantly lower than thechallenge control group (p<0.0001). There were no statisticaldifferences between vaccine titer groups (p≧0.1484) for total lunglesion scores. The negative control group had a median total lung lesionscore of 0.00%.

On study Days 31, 35 and 38 of the post-challenge period, the threevaccine titer groups had significantly less viremia than the challengecontrol group (p≦0.0093). There were no statistical differences betweenvaccine titer groups for viremia post-challenge except on D35, when thehigh titer vaccine group exhibited significantly lower viremia than themedium titer vaccine group (p=0.0442). Negative control piglets werenegative for viremia on D31, D35 and D38.

Clinically, severity (p≦0.0082) and frequency (p≦0.0268) of coughing wasless severe in the three vaccine titer groups than the challenge controlgroup during the post-challenge period; Day 29-Day 38. Pyrexia was moreprominent in the challenge control group than in the three vaccine titergroups post-challenge. ADWG was significantly higher for the threevaccine titer groups compared with the challenge control group(p≦0.0027).

The MID of PRRS 94881 MLV as determined in this study is associated withthe low titer vaccine level of 1×10^(2.77) TCID₅₀/mL, based on arelevant reduction in gross lung lesions for all three titer levels incomparison to the challenge controls after receiving a virulentheterologous European-derived PRRS challenge. When secondary parameterswere examined, all three vaccine titer levels were associated withefficacy and no clear distinctions between titer groups were evident.

General Design of Study:

This was a blinded randomized design study conducted in 70 weaned, PRRSsusceptible piglets, 14-16 days of age on Day 0 (D0). A description oftreatment groups is shown below in Table 2.1:

TABLE 2.1 Treatment Groups No. of Animals Group on D0 Treatment on Day 01 15 IVP No. 1 (mean titer of 1 × 10^(2.77) of PRRS 94881 MLV) 2 15 IVPNo. 2 (mean titer of 1 × 10^(4.42) of PRRS 94881 MLV) 3 15 IVP No. 3(mean titer of 1 × 10^(5.84) of PRRS 94881 MLV) 4 15 CP (Placebo matchedproduct without PRRS 94881 MLV) 5 10 CP (Placebo matched product withoutPRRS 94881 MLV)

Eighty-three piglets met the study inclusion criteria, of which thefirst 70 numerical numbers were randomly assigned to one of five groupson D−3 by a biostatistician. Piglets were assigned 15 per group toGroups 1-4 and ten piglets to Group 5. All 83 piglets were PRRSseronegative.

Piglets were observed from D−1 to D26 for clinical assessmentspost-vaccination and observations will be recorded on the ClinicalAssessment Record form.

Serology:

Venous whole blood was collected from piglets on D0, D7, D14, D21, D28.Sample collections were recorded. Blood samples were spun down and serumwas harvested from each tube, split and transferred to appropriatelylabeled tubes. One set of serum samples were held at 2-8° C. and theother set of serum samples were held at −70±10° C. The set of serumsamples collected on days 0, 7, 14, 21, 28 and 38 and held at 2-8° C.were tested for PRRS antibodies. Results were reported as negative(ELISA S/P ratio of <0.4) or positive (ELISA S/P ratio of ≧0.4).

PRRS Viremia:

The set of serum samples collected on days 0, 7, 14, 21, 28, 31, 35, and38 and held at −70±10° C. were tested for PRRSv RNA by qPCR (Addendum 1,Attachment 7). Results were reported as n.d. (not detected), positive(EU PRRSv detected, but not quantifiable, GE/mL (genome equivalent)=<3.3log) or a reported value (log GE/mL). For statistical purposes, “notdetected” was assigned a value of 0 log GE/mL and a “positive” value wasassigned a value of 3.0 log GE/mL.

Average Daily Weight Gain (ADWG):

Each pig was weighed on a calibrated scale and individual body weightswere recorded. The average daily gain was determined from the D0 to D28and from D28 to D38.

Clinical Observations Post-Challenge:

Piglets were observed by the Study Investigator or designees forclinical signs of disease from D27 to D38 and were recorded on theClinical Observation Record form. Observations included respiration,behavior and cough based on the clinical observation scoring system asshown below in Table 2.2

TABLE 2.2 Clinical Observation Scoring System Respiration Score BehaviorScore Cough Score 0 = normal respiration 0 = normal 0 = no coughing 1 =panting/rapid 1 = mild to moderate 1 = soft or intermittent respirationlethargy cough 2 = dyspnea 2 = severely lethargic 2 = harsh or severe, 3= dead or recumbent repetitive cough 3 = dead 3 = dead

A daily total clinical observation score for each piglet was determinedby the summation of its daily respiration, behavior and cough scores.

Rectal temperatures were collected from D27 to D38.

Total Lung Lesion Score:

All piglets that died before D38 and remaining piglets that wereeuthanized on D38 were necropsied. Each set of lungs was examined forany gross lung pathology and determination of the % pathology for eachlung lobe. If pathology of other organs were noted, these were describedand noted as well.

Lung qPCR for PRRSV:

For each set of lungs, two samples from the Left and Right Apical lobes,the Left and Right Cardiac lobes, the Left and Right Diaphragmatic lobesand the Intermediate lobe, were retained. For one set of lung samples,all three samples from the left side were combined into one container;while all three samples from the right side and the Intermediate lunglobe sample were combined into another container. Each container wasfilled with a sufficient amount of 10% formalin solution. For the otherset of lung samples, all three lung samples from the left side werecombined into one WHIRLPAK®; while all three samples from the right sideand the Intermediate lung lobe sample were combined into anotherWHIRLPAK®.

Frozen lung tissue samples were held at −70±10° C. until furtheranalysis. For each piglet, all left lung samples were homogenized andtested as a single combined sample; and all right lung tissues and theintermediate lung lobe sample were homogenized and tested as a singlecombined sample. Results were reported as n.d. (not detected), positive(EU PRRSv detected, but not quantifiable, GE/mL (genome equivalent)=<3.3log) or a test value (log GE/mL) for left and right lung samples. Foranalysis purposes for each piglet, the mean of left and right lungsample qPCR results were noted. For statistical purposes, “not detected”was assigned a value of 0 log GE/mL and a “positive” value was assigneda value of 3.0 log GE/mL.

Results

Total Lung Lesion Score Post-Challenge:

A summary of group minimum, maximum, median, 95% confidence interval, Qrange and mean for total lung lesion scores showed that the low, mediumand high titer vaccine groups had median total lung lesion scores of0.13%, 0.55%, and 0.40%, respectively; while the challenge control grouphad a median total lung lesion score of 33.40%. The median total lunglesion scores for the three vaccine titer groups were significantlylower than the challenge control group (p<0.0001). There were nostatistical differences between vaccine titer groups (p≧0.1484) fortotal lung lesion scores. The negative control group had a median totallung lesion score of 0.00%.

For one of the animals (high titer vaccine group), histologically mildsuppurative interstitial pneumonia with copious fibrinopurulentpleuritis was noted. Airways and alveoli were relatively unremarkableexcept for scattered neutrophils. Lung tissue was IHC negative for M.hyo, PCV2, PRRSv and SIV antigens. Lung lesions were consistent withserositis generally associated with bacterial agents (Addendum 12;Accession 2009030254). Several pure bacterial cultures from lung tissuewere isolated and identified as Bordetella bronchiseptica and acoagulase negative Staphylococcus. Although two types of bacteria wereisolated from lung tissues, this piglet was not removed from Group 3total lung lesion score analyses.

Two of 10 negative control piglets exhibited very minor lung lesions(No. 1767, 0.55%; No. 1789, 0.61%). These lesions were consideredinsignificant and not indicative of PRRS. Two of 10 negative controlpiglets exhibited very minor lung lesions (No. 1767, 0.55%; No. 1789,0.61%). These lesions were considered insignificant and not indicativeof PRRS.

PRRS Viremia Post-Challenge:

Individual PRRS viremia post-challenge results (D31-D38) were tabulatedand it was found that all piglets were viremic post-challenge in thethree vaccine titer groups and the challenge control group. At all threetime points post-challenge, the three vaccine titer groups hadsignificantly less viremia than the challenge control group (p≦0.0093).There were no differences between vaccine titer groups for viremiapost-challenge except on D35, when the high titer vaccine groupexhibited a lower mean viremia than the medium titer vaccine group(p=0.0442). Negative control piglets were negative for viremia on D31,D35 and D38. Area under curve (AUC) represents both the quantity andduration of viral load and is a good assessment tool for examiningviremia. Significant differences were also detected between the threevaccine titer groups and the challenge control group with respect to AUCat both D28 to D38 (p≦0.0162), and D31 to D38 (p<0.0001). No differenceswere detected between vaccine titer groups with respect to AUC(p≧0.3669) at both time intervals.

The group frequency of viremic positive piglets was also summarized forD31 to D28 and is shown in Table 2.3. Since all vaccine and challengecontrol piglets were viremia positive post-challenge, the frequencyviremia positive piglets was 100% for each group at each time point.Hence, no analyses were conducted on post-challenge frequency forviremia.

TABLE 2.3 Summary of Group Frequency of Viremic Positive Piglets - D 31to D 38 No. Total Study Day Group* Positive % Positive 95% CI No. 31 114 100 76.8 100.0 14 2 15 100 78.2 100.0 15 3 15 100 78.2 100.0 15 4 14100 76.8 100.0 14 5 0 0 0.0 30.8 10 35 1 14 100 76.8 100.0 14 2 15 10078.2 100.0 15 3 15 100 78.2 100.0 15 4 14 100 76.8 100.0 14 5 0 0 0.030.8 10 38 1 14 100 76.8 100.0 14 2 15 100 78.2 100.0 15 3 15 100 78.2100.0 15 4 14 100 76.8 100.0 14 5 0 0 0.0 30.8 10 *Group 1 = Low titerPRRS 94881 MLV; Group 2 = Medium titer PRRS 94881 MLV; Group 3 = Hightiter PRRS MLV; Group 4 = Challenge control group; Group 5 = Negativecontrol group

Lung qPCR Results:

Individual lung virus isolation results post-challenge were summarizedas was the frequency of qPCR positive lung sample test results(p-values) for differences between groups. Lung tissues from piglets inall three vaccine titer groups and the challenge control group were qPCRpositive for PRRSv post-challenge. There were no significant differencesdetected between vaccine titer groups and the challenge control group(p=1.0000). Since all vaccine titer piglets were qPCR positive forPRRSv, no tests were conducted between vaccine titer groups.

Although no differences were detected between vaccine titer groups andthe challenge control group for frequency of qPCR positive lung tissues,differences were evident for viral load in lung tissues. Indeed, thelow, medium and high titer vaccine groups had median lung qPCR values of6.88, 6.80 and 6.81 log₁₀ GE/mL, respectively; while the challengecontrol group had a median lung qPCR value of 8.13 log₁₀ GE/mL. Thedifferences between the vaccine titer groups and the challenge controlgroup were significant (p≦0.0001). Conversely, no differences weredetected between vaccine titer groups for median lung qPCR values(p≧0.7379).

Clinical Observation Scores Post-Challenge:

Abnormal respiration and behavior were not severe post-challenge, asevidenced by median maximum clinical scores of 0 (a score of 0represented normal respiration or normal behavior) for all five groups.In addition, no significant differences were detected between vaccinetiter groups and the challenge control group for both abnormalrespiration and behavior (p≧0.0996).

Coughing was noted in all three vaccine titer groups and the challengecontrol, but was more severe in the challenge control group. For thethree vaccine titer groups, each group had a maximum cough score of 1,which represented soft or intermittent coughing and median maximum coughscore of 0. Conversely, the challenge control group had a maximum coughscore of 2, which represented harsh or severe, repetitive coughing, anda median maximum cough score of 1. The three vaccine titer groups hadsignificantly less severe coughing than the challenge control group(p≦0.0082). Coughing was not noted in the negative control group.

All three vaccine titer groups had maximum total scores of 1 and medianmaximum scores of zero. Conversely, the challenge control group had amaximum total score of 4 and a median maximum score of 1. The threevaccine groups had significantly lower maximum total clinical scoresthan the challenge control group (p≦0.0047). Again, the negative controlgroup had a maximum total clinical score of zero and a median maximumtotal clinical score of zero.

The frequency of abnormal respiration or behavior for at least one dayfrom D29 to D38 was low for all groups. Indeed, no abnormal respirationwas noted in low and medium vaccine titer groups from D29 to D38. Thehigh titer vaccine group had one of 15 (7%) piglets and the challengecontrol group had 3 of 14 (21%) piglets with abnormal respiration.Abnormal behavior was not noted in any vaccine titer group; while 2 of14 (14%) challenge control piglets exhibited abnormal behavior for atleast one day post-challenge. No significant differences were detectedbetween vaccine titer groups and the challenge control group forfrequency of abnormal respiration or behavior for at least one daypost-challenge (p≧0.0996). Abnormal respiration or behavior was notnoted in the negative control group.

The frequency of coughing was much higher in the challenge control thanin the three vaccine titer groups. Indeed, the frequency of coughing forat least one day post-challenge was 14%, 13% and 27% for the low, mediumand high titer vaccine groups, respectively. Conversely, the frequencyof coughing for at least one day post-challenge for the challengecontrol group was 71%. The three vaccine titer groups had significantlyless frequency of coughing than the challenge control group (p≦0.0268).

The frequency of any clinical sign post-challenge, as represented by atotal clinical score >0, was higher in the challenge control group thanin the three vaccine titer groups. Similar to the frequency of cough,the frequency of any clinical sign for at least one day post challengewas 14%, 13%, and 33% for the low, medium, and high titer vaccinegroups, respectively; while 79% of piglets in the challenge controlgroup had a least one clinical sign post-challenge. The three vaccinegroups had significantly lower frequency of any clinical signpost-challenge than the challenge control group (p≦0.0253). No clinicalsigns were noted in the negative control group during this same timeperiod.

Mean scores for abnormal respiration or behavior from D29 to D38 werelow for all groups. Indeed, the mean respiration scores for the low andmedium vaccine titer groups were 0.00 (normal), the high titer vaccinehad a mean respiration score of 0.01 and the challenge control had amean respiration score of 0.03. The mean score for behavior was 0.00 forall three vaccine titer groups; while the challenge control had a meanbehavior score of 0.01. No significant differences were detected betweenvaccine titer groups and the challenge control group for meanrespiration and behavior scores (p≧0.0996). Respiration and behaviormean scores for the negative control group were 0.00.

The challenge control group had a higher mean cough score than the threevaccine titer groups. Indeed, the mean cough scores were 0.01, 0.01 and0.04 for the low, medium and high titer vaccine groups, respectively.Conversely, the mean cough score for the challenge control group was0.28. The three vaccine titer groups had significantly lower mean coughscores than the challenge control group (p≦0.0077).

The mean total score was higher in the challenge control group than inthe three vaccine titer groups. Similar to the mean cough scores, themean total scores post challenge were 0.01, 0.01, and 0.04 for the low,medium, and high titer vaccine groups, respectively; while the meantotal score for the challenge control group was 0.32. The three vaccinegroups had significantly lower mean total scores than the challengecontrol group (p≦0.0025).

Rectal Temperatures Post-Challenge:

The maximum group mean rectal temperatures for low, medium, and hightiter vaccine groups between D29 to D38 were 40.20° C. (D33), 40.33° C.(D35), and 40.20° C. (D37), respectively. The maximum group mean rectaltemperature for the challenge control group and the negative controlgroup between D29 and D38 were 40.51° C. (D33) and 39.95° C. (D33),respectively.

The low titer vaccine group had significantly lower rectal temperaturesthan the challenge control group on D29 (39.47 vs. 39.90° C.), D31(39.85 vs. 40.20° C.), D35 (39.80 vs. 40.22° C.) and D38 (39.86 vs.40.32° C.) (p≦0.0317); while the low titer vaccine had a significantlyhigher rectal temperature than the challenge control group on D30 (40.08vs. 39.58° C.; p=0.0003). No significant differences were detectedbetween the low titer vaccine group and the challenge control group onD32-D34 and D36-D37 (p≧0.0545).

The medium titer vaccine group had significantly lower rectaltemperatures than the challenge control group on D31 (39.62 vs. 40.20°C.), D33 (40.15 vs. 40.51° C.), and D38 (39.58 vs. 40.32° C.)(p≦0.0227). No significant differences were detected between the mediumtiter vaccine group and the challenge control group on D29-D30, D32, andD34-D37 (p≧0.0580).

The high titer vaccine group had a significantly lower rectaltemperature than the challenge control group on D33 (40.12 vs. 40.51°C.), D35 (39.79 vs. 40.22° C.), and D38 (39.55 vs. 40.32° C.)(p≦0.0147); while the high titer vaccine group had a significantlyhigher rectal temperature than the challenge control group on D32 (40.31vs. 39.90° C.; p=0.0063). No significant differences were detectedbetween the high titer vaccine group the challenge control group onD29-D31, D34 and D36-37 (p≧0.0708).

There was less frequency of pyrexia in the three vaccine titer groupspost-challenge compared with the challenge control group. The frequencyof pyrexia was low overall and similar between vaccine titer groups.

Average Daily Weight Gain (ADWG):

The least square mean ADWG from D0 to D28 for the low, medium and hightiter vaccine groups were 0.4, 0.3, and 0.4 kg/day, respectively. Theleast square mean ADWG during this same time period for the challengecontrol group was 0.3 kg/day. The low titer vaccine group had asignificantly higher least square mean ADWG than the challenge controlgroup from D0 to D28 (p=0.0292); while no other significant differenceswere detected between vaccine titer groups and the challenge controlgroup (p≧0.1262), or between vaccine titer groups (p≧0.1293), for leastsquare mean ADWG. During this same time period, the negative controlgroup had mean ADWG of 0.5 kg/day.

The least square mean ADWG from D28 to D38 for the low, medium and hightiter vaccine groups were 0.5, 0.5 and 0.4 kg/day, respectively. Theleast square mean ADWG during this same time period for the challengecontrol group was 0.3 kg/day. The three vaccine titer groupssignificantly out gained the challenge control group post-challenge(p≦0.0027). During this same time period, the negative control group hada mean ADWG of 0.6 kg/day.

Clinical Assessments Post-Vaccination:

In the low titer vaccine group, one piglet (1735) was noted as thin fromD0 to D10. In addition, one piglet beginning on D6 was noted as thin for16 days, exhibited cough for 2 days and depression for 9 days, and waseuthanized on D21 for animal welfare reasons due to poor health. Piglet1727 had a total lung lesion score of 10.8%. Since this value wasdetermined pre-challenge, it was not included in post-challenge totallung lesion analysis. In addition, areas of red/purple consolidation inthe cranioventral areas of the lungs were noted, the liver was pale andkidneys had multiple red/purple areas in the renal pelvis. Thepathologist noted fatty infiltration in central lobules in the liver,commonly seen in cases of negative energy balance and consequentlipolysis. No other lesions were observed in sections of liver, kidneyand lung. Lung tissue was positive for EU PRRS by PCR. No growth wasdetected for routine bacterial culture.

In the medium titer vaccine group, one piglet was excluded from thestudy on D0 prior to treatment due to poor health and was replaced byanother piglet. Two Piglets exhibited coughing for one and three days,respectively, beginning on D12. Four piglets were noted as thin on D2,D3 or both D2 and D3.

In the high titer vaccine group, one piglet (1728) exhibited lameness orlameness and swelling in one leg from D7 to D26. Beginning 18 dayspost-vaccination, one piglet was noted as thin for 6 days, and exhibitedcoughing for one day and rough hair coat for 4 days. Another Piglet wasnoted as thin on D2. Two Piglets exhibited coughing for two days and oneday, respectively, beginning on D9. One piglet exhibited diarrhea forone day (D14).

In the challenge control group, six piglets exhibited periodic coughingfor a cumulative of one to six days, beginning with a first piglet on D7and ending with three piglets on D21. Two piglets were noted as thin fortwo and 11 days, respectively, beginning on D1 for one of these piglets.The second of these two Piglets also exhibited depression and rough haircoat for 4 days, weak on legs for one day and was found dead on D15. Atnecropsy for this piglet there were no lung lesions (lung lesion scoreof 0%) noted, no feed in the stomach and no abdominal fat, and diagnosedstarvation as the cause of death. Since this lung lesion score wasdetermined pre-challenge, it was not included in post-challenge lunglesion analysis.

The group test results (p-values) were summarized for any abnormalclinical assessment for at least one day from D1 to D26. No significantdifferences were detected between vaccine titer groups and the challengecontrol group (p≧0.0502); nor between vaccine titer groups (p≧0.3898).No piglets in the negative control group exhibited an abnormal clinicalassessment from D−1 to D26.

PRRS Serology:

Individual piglet PRRS ELISA serology results were summarized. Pigletsin the negative control group remained PRRS seronegative throughout thestudy. Seroconversion could be observed in the 3 vaccine titer groups by14 days post-vaccination; while the challenge control group remainedPRRS seronegative until post-challenge. Ten days post-challenge (D38)all piglets in the low and high titer vaccine groups and the challengecontrol group were PRRS seropositive; while 14 of 15 piglets in themedium titer vaccine groups were PRRS seropositive.

PRRS ELISA positive serology test results (p-values) were determined fordifferences between groups. The three vaccine titer groups hadsignificantly higher frequencies of PRRS ELISA positive piglets than thechallenge control group on D14, D21 and D28 (p<0.0001). No significantdifferences were detected between the three vaccine titer groups andchallenge control on D38 (p=1.0000 or no test conducted) nor between thethree vaccine titer groups at any time point (p=1.0000 or no testconducted).

PRRS Viremia Post-Vaccination:

Individual PRRS viremia results post-vaccination were determined. Forstatistical purposes, a “not detected” result was assigned a value of 0log GE/mL and a “positive” value was assigned a value of 3.0 log GE/mL.All groups were PRRS viremia negative on D0. The group viremia (qPCR)—D7to D28 post-vaccination titer (log GE/mL) data was assessed. Piglets inthe three vaccine titer groups reached peak mean viremia on D7, afterwhich titers levels for all three groups slowly declined prior tochallenge (SD28). Conversely, the challenge control group and thenegative control group remained negative for viremia during thepre-challenge phase of the study. From the group test results (p-values)for D7, D14, D21 and D28 qPCR results it was seen that all three vaccinetiter groups had significantly higher median qPCR values than thechallenge control group for D7-D28 (p≦0.0159). The medium vaccine titergroup had a significantly higher qPCR median value than the low titervaccine group (p=0.0193); otherwise, no differences were detected forqPCR median values between vaccine groups pre-challenge (p≧0.0594).

Four weeks post-vaccination, the frequency of viremia was low in thethree vaccine titer groups.

The following conclusions can be made based upon these study results:

-   -   The absence of any break of biosecurity during the study and the        confirmation of piglet's susceptibility to PRRS confirmed the        validation of the study and its suitability for interpretation    -   Substantial PRRS clinical disease was evident in the challenge        control group, thus validating this challenge model as an        adequate laboratory tool to evaluate PRRS vaccine efficacy and        more specifically, the MID of PRRS 94881 MLV    -   All three dose levels of PRRS 94881 MLV were associated with        significant reduction in lung lesions, as well as significant        reduction in viremia post-challenge, viral load in lung tissues,        coughing, total clinical observation scores, pyrexia and ADWG    -   The MID of PRRS 94881 MLV as determined in this study is        associated with the low titer vaccine level of 1×10^(2.77)        TCID₅₀/mL, based on a relevant reduction in gross lung lesions        for all three titer levels in comparison to the challenge        controls after receiving a virulent heterologous        European-derived PRRS challenge.

Further Depiction of Results

Clinical observations were taken every day. Quantitative RT-PCR wasperformed using PRRSV European specific primers for samples form blood,oral, fecal, and nasal swab, as well as lung lavages.

From these studies data showed that the piglets showed normal healthexcept for a few pigs that were lame. Post-mortem there were noabnormalities at necropsy except that 1-2 animal showed signs of mildlyenlarged inguinal lymph nodes. Importantly, it was seen that there wereno lung lesions observed with the vaccinated group.

FIG. 8 shows the percentage of viremic animals in the sentinel group ascompared to the group vaccinated with a composition containing theattenuated PRRS virus strain deposited at ECACC Accession No. 11012502.This Figure shows the spread of the vaccine strain from vaccinatedanimals to sentinels. At the peak of PRRS viremia (SD21) as detected byquantitative RT-PCR, the viral load was 78.47% lower in sentinelinfected pigs (mean viral load of 3.347 GE/ml) than in vaccinatedanimals (mean viral load of 4.014 GE/ml). In the room where the PRRSnaïve sows were commingled with their vaccinated offspring, only 3 sowsout of 8 were tested positive for PRRSV in blood by RT-PCR thusconfirming limited and ineffective PRRS MLV vaccine exposure to naïveadult animals. The vaccine virus 94881 MLV was primarily excreted in thefeces in this study. Indeed, in feces, the virus could be detected fromone day to 21 days post vaccination. At five days post vaccination,almost 30% of the vaccinated animals excreted virus in feces. The PRRSvirus was not detected in nasal secretions and in only a few animals viaoral secretions (2 out of the 56 sampled animals at 5 days postvaccination).

Example 3 Exemplary Materials and Methods for Use in Testing VaccineEfficacy Using PORCILIS® PRRS as an Example

A selected number, for example, fourteen healthy pregnant sows from aconfirmed PRRSV negative herd (tested virologically and serologically)were used in this study. Sows faced first or second parturition and wereconfirmed to be pregnant at the time of vaccination/challenge infectionon day 94 of gestation. The sows were divided into three treatmentgroups. The first group was treated with a commercial dose of Porcilis™PRRS of 2 ml containing at least 10⁴° TCID₅₀ via intramuscularadministration at day 94 of gestation. The challenge control group(group 2) received a dose of 10^(4.72) TCID₅₀ in 2 ml cell culturemedium of the pathogenic European field isolate (passage 4)intranasally. Group 3 was vaccinated with a dose of 2 ml containing10^(7.6)TCID₅₀ i.m. PRRS MLV containing attenuated PRRS strain depositedat ECACC Accession No. 11012502 on Jan. 25, 2011 seven days beforeinsemination and was challenged with the European field isolate (passage4) (10^(4.72) TCID₅₀ in 2 ml cell culture medium i.n.) at day 94 ofgestation.

Animals from group 1 were monitored until day 5 post-farrowing. Animalsfrom group 2 and 3 were monitored until day 28 post farrowing.

Animal Phase: All sows were accustomed to the animal facilities 1 weekbefore vaccination. Sows and piglets were observed for their generalhealth status by the investigator on a daily basis. Every animal thatdied or was euthanized was subjected to post-mortem examination andsubsequent laboratory analysis.

Pregnancy was confirmed with ultrasound examination. Serum from sows wasobtained on study days 0, 7, 14 and at farrowing for PCR and ELISAinvestigations. Any material that was associated with abortion wassubjected to laboratory investigations.

Routine gross pathology was performed on all deadborn piglets. Lungtissue samples from all lung lobes were collected from deadborn pigletsand from mummies. Samples for PCR testing were stored at −70° C. 2 ml ofprecolostral blood from each piglet was collected on the day of birth.Serum was prepared and aliquots were stored at −70° C. Serum was used totest for viremia to evaluate the transplacental infection. All pigletsof group 1 that survived until day 5 were euthanized at 5 days of age.

Clinical and Reproductive Performance Parameters: The following criteriaare exemplary criteria that may be investigated (priority order): numberof live born piglets per litter, number of stillborn piglets per litter,number of mummified fetuses per litter and number of piglets survivingthrough day 5 or 28 of age, respectively. The number of piglets bornviremic was determined using pre-colostral serum. The frequency of PCRpositive blood and tissue samples from sows and/or piglets wasinvestigated to evaluate the epidemiology and course of infection.

Field Samples: The field samples investigated in this study were takenfrom routine PRRSV diagnostics and consisted of blood, serum and variousorgan materials, mostly lungs and lymph nodes, from different Europeancountries. The samples were stored at −20° C. for a maximum of 3 daysbefore RNA preparation and residual material was subsequentlytransferred to −70° C. for long term storage. RNA and RT-PCR productswere stored at −20° C.

Cell Culturing: MA104 cells (clone CL2621) were grown in MEM (Dulbecco,Germany) supplemented with 10% FCS and antibiotics.

Porcine alveolar macrophages were harvested using a method described byWensvoort et al. (Wensvoort, G. et al. Vet. Quat. 1991, 13:121-130) andmodified as follows: each lung lobe was infused with 50-100 ml PBS andsubsequently massaged for 3 to 5 min. Then the fluid was rescued fromthe lobe and passed through a gaze filter. This procedure was repeateduntil the lavage fluid was clear. The pooled lavage fluid wascentrifuged at 500 g for 15 min at room temperature. The pellet waswashed in PBS and aliquots of 1×10⁷ cells in 50% RPMI 1640 (Biochrom),40% FCS and 10% DMSO were frozen at −196° C. For further use the PAMswere cultured in RPMI 1640 medium supplemented with 10% FCS andantibiotics.

Preparation of Organ Material for Virus Isolation in Cell Culture: About0.5 cm³ of tissue material was transferred into a tube containing onesteel homogenizer ballet in 1.8 ml of sterile PBS. The tubes wereagitated for 10 min until the organ material was homogenized. Celldebris was pelleted by centrifugation for 2 min at 450 g and roomtemperature. The supernatant was passed through a 0.45. mu.m poresterile filter and stored at −70° C. Aliquots of 30 μl were used toinoculate one semiconfluent cell culture monolayer using 24 wellmicrotiter plates.

RNA Isolation: RNA from organ material was extracted with the RNeasyMini Kit and from serum, plasma, cell culture supernatant and vaccinesolution with the QTAamp Viral RNA Mini Kit (both Qiagen) according tothe manufacturer's recommendations, using approximately 100 mg organmaterial and 140 μl fluid material, respectively, for each preparation.The RNA was finally eluted in 65 μl buffer as recommended by themanufacturer.

Plaque Purification of Virus: Confluent monolayers of Ma104 cells incell culture dishes of 10 cm seeded 48 hours before were infected withthe respective virus at tenfold dilutions from 10⁻¹ to 10⁻⁴. The cellswere incubated for 1 hour with the virus dilutions which were thenremoved, and the cells were overlaid with 30 ml of Ma104 mediumcontaining 5% methylcellulose (Sigma). Plaques were picked after five toseven days and were transferred to Ma104 monolayers in 24 well plates.Virus from these plates was harvested at about 50% CPE and was subjectedto further analysis.

Immunofluorescence Assay: Cells were fixed at −20° C. for 15 min usingice-cold aceton:methanol (1:1) and air-dried thereafter. Afterrehydration in PBS, cells were incubated with the PRRSV specificmonoclonal antibody SDOW17 (Rural Technologies Inc., USA) diluted 1:1000in PBS for 1 hour. After 3 washes with PBS, cells were incubated withgoat anti-mouse FITC conjugated secondary antibody (Dianova, Hamburg,Germany) (1:150 in PBS) for another hour. After 3 final washes with PBS,cells were overlaid with glycerine:PBS solution (1:1) and subjected toimmunofluorescence microscopy.

Diagnostic nRT-PCR: A diagnostic RT-nPCR can be carried out to check thesamples for the presence of PRRSV-EU virus.

An exemplary diagnostic RT-nPCR can be carried out with the Titan OneTube Kit (Roche Molecular Biochemicals) as follows: [5 μl total RNApreparation, 1*RT-PCR buffer, 0.4 mM dNTPs, 20 pmol of primers PLS andPLR, 5 mM dithiothreitol, 1 mM MgCl₂, 2.5-5 U RNasin (Promega Ltd),1-2.5 U enzyme mixture, adjusted to a final volume of 25 μl with DEPCtreated aqua dest]. Routine cycling conditions used can be: 45° C. for 1hour, 94° C. for 2 min and 30 cycles of 94° C. for 30 sec, 58° C. for 45sec and 68° C. for 45 sec, final elongation step at 68° C. for 5 min.The nested PCR reaction was carried out with Qiagen Taq (Qiagen AG) asfollows: [1 μl RT-PCR product, 1*PCR buffer, 10 μl Q-solution, 3.5 mMMgCl₂, 0.3 mM dNTPs, 20 pmol of each EU-7-n-s and EU-7-n-as primers, 2.5U Taq polymerase, adjusted to a final volume of 50 μl with aqua dest].Cycling conditions were as follows: 7 cycles with 94° C. for 1 min, 58°C. for 1 min and 72° C. for 1 min, followed by 30 cycles with 94° C. for1 min and 70° C. for 1.5 min (no annealing step), final elongation stepat 70° C. for 5 min.

Nucleotide Sequencing: Nucleotide sequencing can be performed on thenested PCR products which had been generated with primers that containedan M13 tag, either directly from the PCR reaction or from PCR productsthat had been excised from agarose gels and purified with the JETsorbgel extraction kit (Genomed). Sequencing was done using the automatedsequencer LI-COR DNA Analyzer GENE READIR 4200® (LI-COR Inc., Lincoln,Nebr., USA). Nucleotide and deduced amino acid sequences can be analyzedwith ALIGNIR®, vs1.1 (LI-COR Inc., Lincoln, Nebr., USA) and the DNASIS®2.6 software package (Hitachi Software Genetic Systems Inc., SanFrancisco, USA).

Example 4 Determination of the Full Length Genome Sequence PRRSV 94881

The present example shows the determination of the full length genomenucleotide sequences of the attenuated 94881 strain and its parentalstrain 94881, passage 5. These sequences did not show any unclearnucleotide position what indicates the presence of a homogeneous viralcontent. The comparison of the 94881 Master Seed Virus with the EuropeanReference Virus strain Lelystad Virus (LV) revealed nucleotidehomologies ranging from 85.40 to 95.09 percent in the 8 different viralgenes and amino acid identities from 86.39 to 97.27 percent between bothvirus strains. Two deletions in the ORF 1a of 94881 MSV could beidentified compared to LV. The comparison between 94881 Master SeedVirus and its parental strain, passage 5, showed 26 nucleotide exchangesbetween both resulting in a total number of 14 amino acid exchanges.

For the full length genome sequence determination of the 94881 MasterSeed Virus a total number of 1 reverse transcription, 17 external PCRsand 58 internal PCRs was performed, resulting in 40 PCR products whichwere used for sequencing. In case of 94881, passage 5, 1 reversetranscription, 17 external PCRs and 67 internal PCRs were performed,resulting in 40 PCR products, also, used for sequencing.

Overlapping sequence alignment analyses resulted in a full lengthsequence of 14843 nucleotides for both virus isolates comprising thecomplete open reading frames (ORFs) 1a to 7, each. Additionally, 177nucleotides of the 5″-non translated region (5′ NTR) and 43 nucleotidesof the 3″-non translated region (3′NTR) could be determined, each.Compared with the European PRRSV reference virus isolate Lelystad (LV)(GenBank Assession no. M96262) 44 nucleotides of the 5′NTR and 83nucleotides of the 3′NTR could not be determined as those regions wereused for primer annealing regions.

The sequencing reactions resulted for both virus strains in a clearnucleotide sequence without any wobbles or any other indication on amixed sequence. After translation into amino acids clear amino acidsequences without any questionable amino acid were available forsequence comparison of 94881 MSV with the LV and with the parentalstrain 94881 passage 5. The alignments and comparisons of the nucleotidesequences between 94881 MSV and Lelystad virus were performed and showedthat there were substantial differences at the nucleotide and amino acidlevel. Alignments also were performed between 94881 MSV and its parentalstrain, passage 5.

Sequence comparisons with LV resulted in nucleotide homologies from85.40 to 95.09 percent in the 8 different viral genes and amino acididentities from 86.39 to 97.27 percent between both virus strains. Twodeletions in the ORF 1a of 94881 MSV can be identified compared to LV.One deletion of 138 nucleotides is located at position 2154 to 2292 ofLV and results in 46 missing amino acids. At position 2686 to 2688afurther triplet is deleted resulting in the missing amino acidPhenylalanine. An arrangement of all nucleotide homologies and aminoacid identities between LV and both 94881 strains is shown in Table 4.1.

TABLE 4.1 Arrangement of nucleotide and amino acid sequence comparisonsof the 94881 Master Seed Virus to the European Reference Virus LelystadVirus no. of genetic no. of amino nucleotide homology amino acid aciddeviations to Lelystad deviations identity length of the viral toLelystad Virus in to Lelystad to Lelystad ORF gene/protein nn/aa Viruspercent Virus Virus in 5′NTR 177*/—   9 94.92 — —  1a 7050**/2349 105085.40 326 86.39  1b 4392/1463  346 92.12  40 97.27 2 750/249  67 91.07 23 90.76 3 798/265  72 90.98  28 89,43 4 552/183  52 90.58  23 87.43 5606/201  58 90.43  23 88.56 6 522/173  26 95.02  5 97.11 7 387/128  1995.09  9 92.97 3′NTR 44***/—   2 95.45 — — NTR: non translated region *=Only 177 nucleotides were compared between Lelystad Virus and 94881 MSV.The remaining 44 nucleotides, located upstream, had not been determined.**= The isolate 94881 MSV shows two deletions in the ORF 1a, one with138 nucleotides and one with 3 nucleotides, respectively. The completenucleotide and amino acid sequences of the Reference virus LV were usedfor the homology and identity calculations, deletions are assessed asdeviations. The length of the corresponding viral gene of LV is 7191nucleotides and the corresponding polyprotein 2396 amino acids,calculations of genetic homology and amino acid identity refer to thenumbers of 7191 nucleotides and 2396 amino acids, respectively. ′= Only44 nucleotides were compared between Lelystad Virus and 94881 MSV. Theremaining 83 nucleotides, located downstream, had not been determined

The sequence comparison of the full length nucleotide sequences of 94881Master Seed Virus and 94881 Parental Strain, passage 5, revealed a totalnumber of 26 nucleotide exchanges between both. The nucleotide exchangeswere distributed as follows: 15 in the ORF 1a, 4 in the ORF 1b, 2 in theORF 2, none in the ORF 3, 1 in the ORF 4, 3 in the ORF 5, 1 in the ORF 6and none in the ORF 7. These nucleotide exchanges resulted in a totalnumber of 14 amino acid exchanges which were distributed as follows: 8in the polyprotein 1a, 1 in the polyprotein 1b, 1 in the glycoprotein 2,none in the glycoprotein 3, 1 in the glycoprotein 4, 2 in theglycoprotein 5 and 1 in the matrix protein. Both strains showed the samedeletions compared to Lelystad Virus in ORF 1a. An arrangement of allnucleotide exchanges and resulting amino acid exchanges including thepositions in the viral genes and corresponding proteins is shown indetail in Table 4.2.

Example 5 Culture of Deposited Virus and MSV

As noted above, PARENT (low passage) 94881 is deposited at EuropeanCollection of Cell Cultures (ECACC) under the Accession Numbers ECACC11012501, 94881 Master Seed Virus (MSV) is deposited at EuropeanCollection of Cell Cultures (ECACC) under the Accession Numbers ECACC11012502. The growth and culture conditions for the parent virus and theMSV are provided in the present example.

Parent 94881:

This is a virus of a genotype which is PRRSV type 1, as such it is aEuropean genotype PRRSV. The virus has a porcine host. The parent virusdeposited at 11012501 was deposited at a titer of 5.81 Log 10 TCID₅₀/mL.Host cells for virus propagation are MA 104 cells. These cells arecultured Minimal Essential Medium (MEM) with 3.7 g/L sodium bicarbonatecontaining 6% irradiated Fetal Bovine Serum at 37±1° C. The cells areplated in T-flasks (75 cm²) with a plating density of 2×10⁴ to 2×10⁵cells/cm² and cultivated for 3 to 7 days until 100% confluent prior topassage. For virus growth, the virus is added to the cells at a MOI of0.001-0.01 in T-flasks. The cells infected are at a confluency of about80-100%, typically 1-3 days post cell planting. The infected cells arecultivated at 37±1° C. for 3-10 days post infection and the virus isthen harvested. The harvest is performed when monolayer cells exhibitapproximately 80-100% cytopathic effect (CPE) at 3-10 days postinfection. The supernatant of infected MA104 tissue cultures (spentmedia+PRRSV from a culture with 80-100% CPE) is harvested and containsthe virus that has been propagated. This supernatant may be stored at−70° C./−80° C. for several months until use. The virus is assessed forTCID₅₀ with Spearman and Kaerber calculation to determine log 10TCID₅₀/mL of sample.

Vaccine (high passage) 94881 Master Seed Virus (MSV): This is a virus ofa genotype which is PRRSV type 1, as such it is a European genotypePRRSV. The virus has a porcine host. The MSV deposited at 11012502 wasdeposited at a titer of 6.43 Log 10 TCID50/mL. Host cells used for thepropagation of the MSV are MA 104 cells. These cells are culturedMinimal Essential Medium (MEM) with 1.4 g/L sodium bicarbonate andcontaining 10% irradiated Fetal Bovine Serum at 36±2° C. The cells areplated in T-flasks (75-150 cm2) or 850 cm2 roller bottles with aplanting density of 1×104 to 1×105 cells/cm2 and cultivated for 3 to 7days until 100% confluent prior to passage. For virus growth, the virusis added to the cells at a MOI of 0.001-0.01 in T-flasks or rollerbottles. The cells infected are at a confluency of about 80-100%,typically 1-3 days post cell planting. The infected cells are cultivatedat 36±2° C. for 3-14 days post infection and the virus is thenharvested. The harvest is performed when monolayer cells exhibitapproximately 80-100% cytopathic effect (CPE) at 3-14 days postinfection. The supernatant of infected MA104 tissue cultures (spentmedia+PRRS from a culture with 80-100% CPE) is harvested and containsthe virus that has been propagated. This supernatant may be stored at2-8° C. for 5-10 days, −70° C. for several months until use. The MSV isassessed for TCID50 with Reed and Muench calculation to determine log 10TCID50/mL of sample.

TABLE 4.2 Arrangement of nucleotide and amino acid sequence comparisonsof the 94881 Master Seed Virus to 94881 Parental strain exchangeexchange (nn) from no. of (aa) from no. of position parental synonymous/deviating position parental deviating in viral strain to non amino inviral strain to ORF nucleotides gene MSV synonymous acids protein MSV 1a15 309 C to T synonymous 8 — — 753 G to T non 251 E to D synonymous 1474G to A non 492 V to I synonymous 1789 G to A non 597 V to I synonymous2094 C to T synonymous — — 2987 T to C non 996 L to S synonymous 3034 Ato G non 1012  T to A synonymous 3065 A to G non 1022  E to G synonymous3736 A to G non 1246  T to A synonymous 3966 C to T synonymous — — 4101T to C synonymous — — 5803 C to T non 1935  L to F synonymous 6354 T toG synonymous — — 6519 C to T synonymous — — 6588 T to C synonymous — —1b 4 591 T to C synonymous 1 — — 1833 C to T synonymous — — 1932 C to Tsynonymous — — 3682 G to A non 1228  V to I synonymous 2 2 13 C to T non1  5 H to Y synonymous 195 C to T synonymous — — 3 0 — — — 0 — — 4 1 529T to C non 1 177 F to L synonymous 5 3 109 A to G non 2  37 N to Dsynonymous 364 C to T non 122 L to F synonymous 570 C to T synonymous —— 6 1 214 C to T non 1  72 L to F synonymous 7 0 — — — 0 — —

Example 6 PRRS 94881 _(ML)V Gilt MID Stud

Summary

The objective of this vaccination-challenge study was to evaluate theminimum immunizing dose (MID) of Porcine Reproductive and RespiratorySyndrome Vaccine European-derived Isolate 94881, Modified Live Virus,Code 19T1.U_(PRRS 94881 MLV) in gilts. Two different titer levels wereadministered to PRRS seronegative gilts approximately 28 dayspre-breeding (Day 0; D0), gilts were challenged with a heterologousEuropean isolate of PRRSv at approximately 90 days of gestation (D118)and gilts were evaluated for the total number of live born piglets orpercentages of live born piglets and weaned piglets at 20 days of age todetermine the MID. At the time of challenge on Day 118 (D118), thechallenge control group consisted of 8 pregnant gilts (Group 1,Placebo), the low titer group consisted of 8 pregnant gilts (Group 2,1×10^(2.43) TCID₅₀/dose), the high titer group consisted of 8 pregnantgilts (Group 3, 1×10^(3.90) TCID₅₀/dose), and the negative control groupconsisted of 5 pregnant gilts (Group 4, Placebo, not challenged).

Both the low titer and the high titer groups were associated with higherpercentages of live piglets per litter at farrowing (P≦0.0455) andhigher percentages and numbers of live piglets per litter at weaning(P≦0.0203) in comparison to the challenge control group.

With regard to supportive efficacy parameters, the high dose group wasassociated with a higher percentage and number of healthy piglets pergilt at farrowing (P≦0.0211), a lower percentage and number of weak andmummified feti (P≦0.0090), a lower percentage of qPCR positive gilts andlower viral load in gilts post-challenge on D125, DOF 0 and DOF+13(P≦0.0155), a lower percentage of piglets per gilt qPCR positive andlower piglet viral load on DOF 0 (P≦0.0030), a lower percentage ofpiglets per gilt with clinical disease (P<0.0001), and higher pigletbody weights on DOF+20 and ADWG (P<0.0013), in comparison with thechallenge control group.

The low dose group was associated with a higher percentage of healthypiglets per gilt at farrowing (P=0.0138), a lower percentage and numberof mummified feti (P≦0.0190), a lower percentage of qPCR positive giltsand lower viral load in gilts post-challenge on D125, D132, DOF 0 andDOF+13 (P≦0.0290), a lower percentage of piglets per gilt qPCR positiveon DOF 0 (P=0.0381), a lower percentage of piglets per gilt withclinical disease (P<0.0001), and higher piglet body weight on DOF+20 andADWG (P<0.0028), in comparison to the challenge control group.

In conclusion, the study objective was met and data from this studyestablishes the MID of PRRS 94881 MLV in gilts as 1×10^(2.43) TCID₅₀/2mL. In addition, this study establishes duration of immunity (DOI) ingilts of approximately 4 months.

Objective(s)/Purpose of the Study

The objective of this vaccination-challenge study was to evaluate theminimum immunizing dose (MID) of Porcine Reproductive and RespiratorySyndrome Vaccine European-derived Isolate 94881, Modified Live Virus,Code 19T1.U_(PRRS 94881 MLV), at two different titer levels (Group 2,low titer; Group 3, high titer), administered to PRRS seronegative giltspre-breeding to provide higher percentages of live born piglets andweaned piglets at 21 days of age, following challenge of gilts with aheterologous European isolate of Porcine Reproductive and RespiratorySyndrome virus (PRRSv) at approximately 90 days of gestation. Theprimary criteria to satisfy this objective was that one or both vaccinegroups must demonstrate relevantly higher percentage or number of liveborn piglets and weaned piglets at 20 days of age (DOF+20), comparedwith the challenge control group (Group 1).

Other parameters analyzed between the vaccine groups and the challengecontrol group included gilt clinical assessments post-vaccination, giltPRRS serology, gilt viremia, gilt clinical observations, piglet viremia,total piglets per litter, healthy live piglets per litter, weak livepiglets per litter, mummies per litter, stillborns per litter,crushed/mortalities piglets per litter, piglet clinical observations,and piglet average daily weight gain (ADWG). These parameters wereanalyzed as supportive parameters and did not serve as primaryparameters to satisfy the study objective.

Schedule of Events

TABLE 6.1 Gilt Schedule of Events Study Day(s) Dates Key Study Event −2or −1 20 Jul. 2010 to 21 Jul. 2010 Health examination −1 to 21 21 Jul.2010 to 12 Aug. 2010 Groups 1-4: Daily Clinical Assessments 0 22 Jul.2010 Groups 1-4: Blood collection; Vaccinated Groups 1 and 4 withControl Product (CP); Vaccinated Group 2 with Investigational VeterinaryProduct (IVP) No. 1 (low titer vaccine group); and vaccinated Group 3with IVP No. 2 (high titer vaccine group) 8 to 21 30 Jul. 2010 to 12Aug. 2010 Groups 1-4: Treated gilts once daily with Matrix ™ tosynchronize estrus cycles 7, 14, 21, 56 29 Jul. 2010, 05 Aug. 2010,Groups 1-4: Blood collection and 84 12 Aug. 2010, 16 Sep. 2010, 14 Oct.2010 22 to 113 13 Aug. 2010 to 12 Nov. 2010 Groups 1-4: ClinicalAssessments at least three times a week 26 to 32 17 Aug. 2010 to 23 Aug.2010 Groups 1-4: Observed for heat detection and bred gilts byartificial insemination (Al) 84 14 Oct. 2010 Groups 1-4: Pregnancy checkby ultrasound 116 to 20 15 Nov. 2010 to 05 Jan. 2011 Groups 1-4:Clinical Observations, days after recorded abortions, stillbirths,mummies, farrowing live piglets, weak born piglets 118 (approx. 17 Nov.2010 Groups 1-4: Blood collection 90 days of Groups 1-3: Challenged withPRRSv gestation) isolate 190136 125,132, 24 Nov. 2010, 01 Dec. 2010,Groups 1-4: Blood collection farrowing/ 03 Dec. 2010 to 16 Dec. 2010,abortion (DOF*), DOF 10 Dec. 2010 to 23 Dec. 2010, +7, DOF + 13 16 Dec.2010 to 29 Dec. 2010 DOF + 20 23 Dec. 2010 to 05 Jan. 2011 Groups 1-4:Blood collection from remaining gilts; Euthanized remaining gilts;Disposal DOF + 20 or 25 Dec. 2010 to 05 Jan. 2011 Groups 1-4: Euthanizedremaining gilts; later Disposal *DOF = Day of Farrowing

TABLE 6.2 Piglet Schedule of Events Study Day(s) Dates Key Study EventDOF 03 Dec. 2010 to All dead piglets: Weighed; Necropsied; 16 Dec. 2010Collect Collected blood/body fluid if possible; lung samples All livepiglets: Weighed; Blood collection (pre-colostral or peri-natal (within12 hours of birth)) DOF +1 to 04 Dec. 2010 to All live piglets: ClinicalObservations DOF +20 05 Jan. 2011 Dead piglets: Weighed; Necropsied;Collected blood/body fluid if possible; Collected lung samples DOF +7 10Dec. 2010 to All live piglets: Blood collection 23 Dec. 2010 DOF +13 16Dec. 2010 to All live piglets: Blood collection 29 Dec. 2010 DOF +20 23Dec. 2010 to All live piglets: Weighed; Blood 05 Jan. 2011 collection;DOF +20 or 25 Dec. 2010 to Group 1-3 piglets: Euthanized later 05 Jan.2011 remaining piglets, Disposal; Group 4 piglets: Assigned to anotherBIVI project

Study Design

TABLE 6.3 Study Design Challenged on D118 with 6.0 mL (2 Number NumbermL/nostril; 2 mL IM) of Treatment on D0 (approximately of of 1 ×10^(6.30) TCID₅₀ Group gilts on D0 28 days prior to breeding) gilts onD118 of PRRSv 190136 1 28 2.0 mL IM of Control Product 16 Yes (Challenge(Placebo matched product control without PRRS 94881 MLV) group) 2 28 2.0mL IM of IVP No. 1 (1 × 16 Yes (Low titer 10^(2.43) TCID₅₀ of PRRS group94881 MLV) 3 28 2.0 mL IM of IVP No. 2 (1 × 16 Yes (High titer 10^(3.90)TCID₅₀ of PRRS group) 94881 MLV) 4 10 2.0 mL IM of Control  5 No(Negative Product control group)

Blinding Criteria

The Study Investigator and designees were blinded with regard to giltsassigned to Groups 1-4. To maintain blinding of the Study Investigatorand designees, a person not collecting data administered the IVPs and CPto assigned gilts on D0. Laboratory personnel were blinded to thetreatment each gilt received while conducting their respective tasks.

Materials

Investigational Veterinary Products (IVP) and Control Product (CP)

TABLE 6.4 Investigational Veterinary Products (IVPs) Generic PorcineReproductive and Respiratory Syndrome, Modified Live Virus Product Name:Isolate: Isolate 94881 Formulation: The Manufacturer's Batch Protocol(MBP) for PRRS 94881 MLV vaccine, Lot 390-005 (cake) is presented inAppendix 1. The MBP for Sterile Carbopol Adjuvanted Diluent, Lot 808-002(diluent) is presented in Section 15.1, Appendix 1. D0 just prior tovaccination, BIVI-Ames reconstituted/diluted PRRS 94881 MLV vaccine, Lot390-005 with Sterile Carbopol Adjuvanted Diluent, Lot 808-002 toformulate the two IVPs. IVP No. 1 was formulated to a targeted titerlevel of approximately 1 × 10^(3.0) TCID₅₀/2 mL and IVP No. 2 wasformulated to a targeted titer level of approximately 1 × 10^(4.5)TCID₅₀/2 mL. An adequate volume of each IVP was formulated forvaccination and testing. IVP Lot/Serial IVP No. 1: N270-142 Nos.: IVPNo. 2: N270-143 Manufacturer: Boehringer Ingelheim Vetmedica, Inc. 2621North Belt Highway St. Joseph, MO 64506 Expiration An expiration date of22 Jul 2010 was assigned to each IVP for study Date: purposes only.Storage Rehydrated/diluted IVP was stored at 2-8° C. or on iceRequirements: Testing: PRRS 94881 MLV, Serial 390-005 and SterileCarbopol Adjuvanted Diluent, Lot 808-002 were tested by BIVI-QC. At thestart and end of the vaccination procedure, BIVI-Ames was contacted.BIVI-Ames tested pre- and post-vaccination aliquots of each IVP forvirus titer in accordance with the PRRSv Titer Procedure (Section 15.1).Test Results: Test results for PRRS 94881 MLV, Serial 390-005 and forSterile Carbopol Adjuvanted Diluent, Lot 808-002 were satisfactory IVPNo. 1 had an average titer of 1 × 10^(2.43) TCID₅₀/2 mL and IVP No. 2had an average titer of 1 × 10^(3.90) TCID₅₀/2 mL IVP Transfer: Twovials containing 35 mL of each IVP were transferred to the study site onDO just prior to vaccination. IVP Retention: A retention sample of eachIVP is currently stored at −70 ± 10° C. at BIVI-Ames until the finalreport has been signed

TABLE 6.6 Control Product (CP) Generic Product Placebo Name:Formulation: BIVI-Production produced lyophilized placebo productcontaining inert material comprised in the vaccine serial without PRRS94881 MLV (Lot N240-191-062409, On D0, BIVI-Ames reconstituted LotN240-191-062409 with Sterile Carbopol Adjuvanted Diluent, Lot 808-002 toformulate the CP, Lot No. 270-141. Manufacturer: Boehringer IngelheimVetmedica, Inc. 2621 North Belt Highway St. Joseph, MO 64506, USA LotNumber: N270-141 Expiry Date: An expiration date of 22 Jul 2010 wasassigned to the CP for study purposes only. Storage Conditions:Lyophilized vaccine: 2-8° C. Rehydrated CP: 2-8° C. or on ice Testing:CP was tested by BIVI-QC for EP sterility in accordance with SpecialOutline No. 96 Test Results: CP was determined to be sterile CPTransfer: Two vials containing 50 mL each of CP were transferred to thestudy site on D0 just prior to vaccination. CP Retention: CP wasformulated for this study only and was not retained.

Challenge Material

TABLE 6.7 Challenge Material Name/number of Porcine Reproductive andRespiratory Syndrome virus isolate Location and date Isolate 190136,Passage 2. Isolate #190136 was obtained from lung of isolation incl.tissue of a new born piglet from a farm showing typical reproductiveclinical symptoms signs of PRRS (abortions in gilts and weakness in newborn piglets) during an outbreak in Lower Saxony, Germany, in April2004. The attending veterinarians submitted the samples to bioScreen(sample arrived on 21 Apr. 2004). Isolate #190136 could directly bepropagated on AK-MA104 cells. Formulation: Challenge virus was thawedand diluted with MEM (Minimum Essential Medium) to a targeted titer ofapproximately 1 × 10⁶ TCID₅₀/3 mL on D118. An adequate volume ofchallenge material was prepared. Two aliquots were removed fromchallenge material. Lot Number: N289-034 Manufacture: BoehringerIngelheim Vetmedica, Inc.-USA Storage conditions Bulk challenge materialwas stored at −70 ± 10° C. Once prepared, diluted challenge material wasmaintained on ice until it was administered. Testing: At the start andend of the challenge procedure, BIVI-Ames was contacted. BIVI-Ameslaboratory personnel tested pre- and post- challenge aliquots for virustiter in accordance with the PRRSv Titer Procedure Test Results: Thechallenge material had a mean titer of 1 × 10^(6.30) TCID₅₀/6 mL doseChallenge material Three vials containing 101 mL each of challengematerial were transfer: transferred to the study site on D118 just priorto administration. Administration 2.0 mL/nostril and 2.0 mL IM in theleft neck (administered to all gilts route in Groups 1, 2 and 3 onD118). Challenge material Challenge material was formulated for thisstudy only and was not retention: retained.

Additional Treatments

MATRIX™ (6.8 mL; Alternogest; Intervet/Schering Plough Animal Health)was administered in each gilt's feed from D8 to D21 to synchronizeestrus cycles.

Oxytocin (VetTek) was administered at parturition to assist gilts withfarrowing, but not for initiation of farrowing. At farrowing, all livepiglets received 1.0 mL iron injection (Phoenix or Durvet), 1M, in theright ham for prevention of iron deficiency anemia shortly after birth.Additionally, all live piglets received gentamicin (SparHawkLaboratories Inc.) as a scour preventative shortly after birth. Alltreatments were recorded on the Biological & Pharmaceutical TreatmentRecord form.

Treatments

Dosing Justification

Each IVP was administered as a 2.0 mL dose to assigned gilts to evaluatethe MID of PRRS 94881 MLV. A 2.0 mL dose of the CP was administered togilts assigned to groups 1 and 4.

Dosing Regimen

On D0, each IVP or CP was administered to each respective gilt IM in theright neck region using a sterile 3.0 mL Luer-lock syringe and a sterile18 g×1 inch (2.54 cm) needle by a person not collecting study data. Thedosing regimen is shown below in Table 6.8.

TABLE 6.8 Dosing Regimen Group Number Treatment Dose/Route Study Day 128 CP 2.0 mL IM D0 2 28 IVP No. 1 2.0 mL IM D0 (low titer dose) 3 28 IVPNo. 2 2.0 mL IM D0 (high titer dose) 4 10 CP 2.0 mL IM D0

Methods and Precautions for Study Personnel

Personnel administering IVPs, the CP and challenge material adhered tosafety precautions and wore personal protective equipment as outlinedfor the specific study site.

Animal Information

Details of Study Animals

TABLE 6.9 Animal Information Source: Wilson Prairie View Farms N5627Highway DD Burlington, WI 53105 Number of gilts: 94 Arrival date: Giltsarrived at the Veterinary Resources, Inc. facilities in two shipments on15 Jul. 2010 (D-7) and 20 Jul. 2011 (D-2). Identification: Individuallyear tagged with unique number Species: Porcine Breed: Commercialcrossbred Gender: Females Age range: Approximately 8 months of age on D0Ownership of test Boehringer Ingelheim Vetmedica, Inc. animals:Physiological status: All gilts were PRRS ELISA seronegative on D0.Gilts selected for assignment to the study were observed by the StudyInvestigator on D-2 or D-1 and determined to be in good health andnutritional status. Pregnancy results: Gilts were checked for pregnancyon D84. Group-Gilt Group 1 (n = 28): 1, 3, 5, 6, 8, Group 2 (n = 28):12, 26, 31, 32, 41, Assignments on D0 11, 15, 18, 19, 34, 35, 39, 40,52, 47, 49, 56, 58, 59, 60, 64, 67, 69, 70, 54, 68, 79, 82, 88, 90, 95,96, 72, 73, 75, 76, 77, 78, 85, 89, 93, 94, 98, 101, 102, 107, 109 and110 100, 103 and 104 Group 3 (n = 28): 2, 7, 14, 22, Group 4 (n = 10):4, 10, 13, 16, 17, 23, 25, 27, 28, 30, 33, 36, 42, 20, 21, 24, 29, and108 46, 48, 51, 53, 57, 61, 62, 65, 66, 80, 84, 86, 91, 92, 105 and 106Group-Gilt Nos. Group 1 (n = 16): 1, 6, 11, 18, Group 2 (n = 16): 12,26, 31, 32, 41, Remaining in the 19, 40, 54, 68, 79, 82, 88, 95, 47, 49,58, 64, 67, 72, 85, 89, 93, Study On D118 96, 102, 98 102 and 107 100,and 104 Group 3 (n = 16): 7, 14, 23, 27, Group 4 (n = 5): 4, 13, 16, 17,and 33, 36, 46, 48, 57, 61, 62, 65, 108 66, 84, 92, and 106

Inclusion/Exclusion Criteria

All gilts enrolled in this study were PRRS ELISA negative, non-bred andwere healthy at the time of vaccination as determined by observation.

Post-Inclusion Removal of Gilts

Five (5)—Group 1 gilts (Nos. 5, 15, 34, 35 and 52), two (2)—Group 2gilts (Nos. 77 and 94), three (3)—Group 3 gilts (Nos. 2, 25, and 30) andone (1)—Group 4 gilt (No. 20) did not display estrus and weresubsequently not bred. These gilts were removed from the study by D47.

Two (2)—Group 1 gilts (Nos. 109 and 110), nine (9)—Group 2 gilts (Nos.56, 59, 60, 69, 73, 75, 76, 78, and 103), four (4)—Group 3 gilts (Nos.22, 28, 51 and 53) and one (1)—Group 4 gilt (No. 21) were removed fromthe study by D89 due to lameness, not pregnant, or late breeding.

The study protocol stated that if >16 pregnant gilts per group forGroups 1-3 were still in the study prior to challenge, extra gilts wouldbe randomly selected for removal from the study; thus leaving 16pregnant gilts per group for Groups 1-3. Five (5)—Group 1 gilts (Nos. 3,8, 39, 90 and 101), one (1)—Group 2 gilt (No. 70) and five (5)—Group 3gilts (Nos. 42, 80, 86, 91, and 105) were removed from the study by D104based on randomizations by the statistician or selection by non-studyperson, reducing the group size to 16 gilts for Groups 1-3.

Due to space limitations, the Study Investigator requested that the sizeof Group 4 be reduced from eight (8) to five (5). The statisticianrandomly selected three (3) gilts (Nos. 10, 24 and 29) for removal fromthe study, which were removed on D109.

Animal Management and Housing

Animal Housing

Low IVP titer gilts were housed in Rooms 1 and 2, and high IVP titergilts were housed in Rooms 3 and 4, in Building CB at VRI-Cambridge fromD−1 to study completion. Gilts assigned to the challenge control andnegative control groups were housed in a single room at VRI-Risdal fromD−1 to D85. On D85, remaining gilts in the challenge control andnegative control groups were moved to VRI-Cambridge. Sixteen(16)—challenge control gilts were housed in Building CB, Rooms 5-8 andeight (8)—negative control gilts were housed in Building CA, Room 12,for the remainder of the study. From D85 onward, each room was identicalin layout with two rows of 4 farrowing crates per row. Each crate heldone gilt and her progeny. Each crate was approximately 5 feet×7 feet insize, was elevated off of the floor, metal rod panels for sides andplastic-mesh for flooring. There was no nose-to-nose contact betweenadjacent crates. The floor of each crate was washed down at least oncedaily to remove excrement and waste. Each room had separate heat andventilation, preventing cross-contamination of air between rooms. Eachroom was cleaned and disinfected prior to use for this study. AnimalServices staff showered and donned clean clothes before entering eachroom.

Treatment group isolation was necessary in this study as it is wellknown within the scientific community that PRRSv readily spreads frompig to pig via various mechanisms including aerosolization. Thisincludes avirulent live PRRS vaccines as these biological productsinclude attenuated virus particles that mimic the characteristics ofvirulent wild-type PRRS without the capability to cause disease. Propermethods were in place to ensure that biosecurity was maintained and thatvaccinated animals did not accidentally cross-contaminatenon-vaccinated, PRRSv naïve negative control animals.

Each room in the facility has fans and heaters to aid in sufficient aircirculation and heating. The ventilation system is separate yetidentical for each room, so air is not shared between rooms. Solid feedwas stored in bags, free from vermin. Water was ad libitum from a welllocated at the animal facility. Gilts were fed a commercially prepared,non-medicated gestation or lactation ration (Heart of Iowa Cooperative,Roland, Iowa) appropriate for their size, age, and condition.

Gilts were in good health and nutritional status before initiation ofthe study as determined by the Study Investigator. During the study, twogilts were observed with mild lameness and one gilt was observed with aswelling in the left neck region. The Study Investigator considered allof these to be non-specific conditions that commonly occur in groups ofgilts housed in confinement. The Study Investigator determined thatconcomitant treatments were not required for any animals during thisstudy.

All gilts and their pigs assigned to Groups 1-3 were disposed of bycommercial incineration after euthanasia. Gilts assigned to Group 4 weredisposed of by rendering after euthanasia. Group 4 piglets were noteuthanized and disposed of, but were assigned to another BIVI project.No food products from animals enrolled in this study entered the humanfood chain.

Assessment of Efficacy

To assess the MID of PRRS 94881 MLV, low titer group (Group 2) and thehigh titer group (Group 3) were challenged on D118 and reproductiveperformance and weaned piglets post-challenge were evaluated. Theprimary criteria to satisfy this objective were that one or both vaccinegroups must demonstrate statistically higher percentage or number oflive born piglets and weaned piglets at 20 days of age (DOF+20),compared with the challenge control group (Group 1).

Other parameters analyzed to support efficacy between vaccine groups andthe challenge control group included gilt clinical assessmentspost-vaccination, gilt PRRS serology, gilt viremia, gilt clinicalobservations post-challenge, piglet viremia at farrowing, total numberof piglets per litter, healthy live piglets per litter, weak livepiglets per litter, mummies per litter, stillborns per litter,crushed/mortalities piglets per litter, piglet clinical observations,and piglet ADWG.

Criteria for a Valid Test

The negative control group (Group 4) was not included in any analyses.The negative control group was included in the study to demonstrate thatsource gilts were PRRS negative at the time that the other three groupswere challenged. Furthermore, the negative control group had to remainPRRS negative until study completion to exclude a possible introductionof a field PRRSv or accidental cross contamination from challengedgroups.

Pre-purchase and D0 serum samples were all required to be negative forPRRS antibodies. Serum samples collected from Groups 1 and 4 up to theday of challenge and from Group 4 until study completion had to be freeof PRRS antibodies for the study to be valid.

Primary Outcome Parameters

The primary efficacy variables for statistical evaluation were livepiglets per gilt at birth (mean number or percentage) and live pigletsper litter at DOF+20 (mean number and percentage).

9.2.1 Percentage of Live Piglets at Birth Per Gilt

Farrowing data was recorded for each gilt during the study. The day offarrowing (DOF) for each gilt was defined as the day that the firstpiglet was born. At the time of farrowing, each piglet was classifiedinto one of five categories listed below in Table 6.10. A live piglet atbirth was defined as any piglet that received an observation rating atbirth as healthy live piglet, weak live piglet or crushed/mortalitypiglet (death due to crushing was confirmed at necropsy as describedbelow). Observations were conducted by the Study Investigator or adesignee and were recorded on the Farrowing/Litter Processing Recordform.

TABLE 6.10 Farrowing Result Categories Term Definition Mummy A mummifiedfetus that is not completely developed and is exhibiting severeautolysis or a completely developed fetus exhibiting a shiny gun metalgreen appearance and with no or very little hair Stillborn Piglet Acompletely developed dead piglet with hair Weak Live Piglet A poor-doingpiglet that cannot nurse or walk Healthy Live Piglet A healthy, nursingpiglet that is able to walk Crushed/Mortality A fully developed pigletthat appears to have died shortly after farrowing due to being crushedby the gilt

Live Piglets Per Litter at DOF+20

Piglets were observed for clinical signs of disease as outlined below inTable 6.11 from DOF+1 to DOF+20. Observations were conducted by theStudy Investigator or designees and were recorded on the ClinicalObservations Record form.

TABLE 6.11 Clinical Observation Scoring System Respiration BehaviorCough 0 = normal respiration 0 = normal 0 = none 1 = panting/rapid 1 =mild to moderate 1 = soft or intermittent respiration lethargy cough 2 =dyspnea 2 = severely lethargic 2 = harsh or severe, 3 = dead orrecumbent repetitive cough 3 = dead 3 = dead

A daily total clinical observation score was determined as a summationof respiration, behavior and cough scores by the statistician using SASprogram. Any piglet receiving a clinical score of zero to eight onDOF+20 was evaluated as alive on DOF+20.

Supportive Parameters

Other parameters analyzed between vaccine groups and the challengecontrol group included gilt clinical assessments post-vaccination, giltPRRS serology, gilt viremia, gilt clinical observations, piglet viremia,total piglets per litter, healthy live piglets per litter, weak livepiglets per litter, mummies per litter, stillborns per litter,crushed/mortalities piglets per litter, piglet clinical observations,and piglet average daily weight gain (ADWG).

Gilt Daily Assessments

All gilts were observed once daily from D−1 to D21 and from D22 to 115at least three times a week for daily assessments post-vaccination bythe Study Investigator or designees. Observations were recorded on theDaily Assessment Record form.

Gilt PRRS Serology

Venous whole blood was collected from gilts prior to purchase and on D0,D7, D14, D21, D56, D84, D118, D125, D132, DOF 0, DOF+7, DOF+13 andDOF+20. Blood collections from gilts at the time of farrowing/abortions(DOF 0) were conducted immediately after farrowing/abortions werecompleted or up to 8 hours post-farrowing/abortion.

Briefly, approximately 10 mL of blood was collected from each gilt intoan appropriate sized Serum Separator Tube(s) (SST). Sample collectionswere recorded on the Sample Collection Record form. Blood in SSTs wasallowed to clot at room temperature. Blood samples collected on weekdayswere delivered to BIVI-Ames on the day of collection. Blood samplescollected on weekends were processed by VRI personnel on the day ofcollection. Serum samples at VRI were held at 2-8° C. At eitherBIVI-Ames or VRI, blood samples were spun down and serum was harvested,split and transferred to appropriate tubes. Each tube was labeled withthe gilt's ID number, the study number, the date of collection, thestudy day and the sample type. Serum samples at VRI were delivered toBIVI-Ames at the earliest convenient time. A completed Specimen DeliveryRecord form was included with each shipment. At BIVI-Ames, one set ofserum samples were held at 2-8° C. and the other set of serum sampleswere held at −70±10° C.

The set of gilt serum samples held at 2-8° C. were tested by BIVI-Amesfor PRRS antibodies. Results were reported as negative (ELISA S/P ratioof <0.4) or positive (ELISA S/P ratio of ≧0.4).

Gilt Clinical Observations Post-Challenge

Gilts were observed for clinical signs of disease from D116 to DOF+20.Observations were conducted by the Study Investigator or designees.Gilts were observed each day for respiration, behavior and cough basedon the clinical observation scoring system outlined above in Table 6.11.

9 Piglet PRRS Viremia

Venous whole blood was collected from piglets on DOF 0, DOF+7, DOF+13and DOF+20, or when a piglet was found dead. Pre-colostral bloodcollection was preferred from newborn piglets, but was not mandatory. Ifpre-colostral blood could not be collected, peri-natal blood within 12hours of farrowing was permissible. Samples not collected before firstsuckling were labeled as “Peri-natal” and kept separately frompre-colostral samples.

Briefly, approximately 2.0 to 2.5 mL of blood was collected from eachlive piglet into an appropriate sized Serum Separator Tube(s) (SST). Aminimum of 5.0 mL of blood was collected from each piglet on DOF+20 justprior to euthanasia. Blood was collected from each mummy or stillbirthor if blood could not be collected from a dead fetus, thoracic orabdominal fluid was collected. Sample collections were recorded on theSample Collection Record form.

Piglet Average Daily Weight Gain

Individual piglets were weighed on DOF 0 and DOF+20, or on the day apiglet was found dead by the Study Investigator or designees. Individualbody weights on DOF 0 were recorded on the Farrowing/Litter ProcessingRecord form and body weights after DOF 0 were recorded on the BodyWeight Record form.

PRRS Virus Quantitation in Lung Tissue

All piglets dead at delivery or dying before DOF+20 were necropsied bythe Study Investigator. Necropsy results and a diagnosis were recordedon the Necropsy Report form. Two lung samples were collected from eachnecropsied piglet. One sample was placed into a separate WHIRLPAK®container and another sample was placed into an appropriate containerfilled with a sufficient volume of 10% formalin. Sample collections wererecorded on the Necropsy Report form.

WHIRLPAKS® and formalin containers were appropriately labeled withanimal number, study number, date of sampling, study day, sample typeand whether the samples were from the left side, right side or both.Samples in WHIRLPAKS® were stored at −70±10° C. and samples in 10%formalin were stored at room temperature until delivered to BIVI-Ames. Acompleted Specimen Delivery Record form was included with each deliveryof samples. At BIVI-Ames, samples in WHIRLPAKS® were stored at −70±10°C. until shipped from BIVI-Ames to Germany, and formalin fixed sampleswere stored at BIVI-Ames at room temperature.

After the study was completed, frozen tissue samples in WHIRLPAKS® wereshipped and tested as described above.

Formalin fixed tissue samples were submitted to ISU VDL within one weekof collection for embedding in paraffin blocks). Tissues in paraffinblocks were returned to BIVI and are currently held by BIVI-Ames at roomtemperature for possible future testing. A decision of whether to retainthese samples or discard them will be made by the Study Sponsor andMonitor after the study report is completed.

Adverse Events

No adverse events attributed to the IVPs were reported during thisstudy. For more information on adverse events, see Section 12.6, GiltAssessments Post-Vaccination.

Statistical Methods

Experimental Unit

Treatment groups had to be housed in separate rooms in this study toavoid transmission of live PRRSv vaccine to non-vaccinated groups.Therefore, room was the experimental unit. However, for the purposes ofthis analysis, possible bias due to confounding “room” and “treatment”effects were ignored, and gilt and her corresponding litter wereanalyzed as the experimental unit.

Randomization

Ninety-four (94) PRRS seronegative gilts from a pool of 107test-eligible gilts were randomly assigned to one of 4 groups prior toD0. Groups 1-3 each consisted of 28 gilts. Group 4 consisted of 10gilts. For Group 1, Nos. 45 and 55 were excluded by the farm managerprior to shipment due to health reasons and were replaced by two extragilts, Nos. 15 and 18, respectively. For Group 3, gilt No. 44 wasexcluded by the farm manager prior to shipment due to health reasons andwas replaced by extra gilt No. 25.

Due to space limitations at the time of challenge, Groups 1-3 wererestricted to 16 gilts per group and Group 5 was restricted to 5-8gilts. On D85, 16 gilts per group were randomly selected to remain inthe study for Groups 1-3. Since Group 4 consisted of 8 gilts on D85,this group was not further reduced by randomization. Afterwards, theStudy Investigator requested that Group 4 be reduced from 8 gilts to 5gilts. On D109, 5 gilts were randomly selected to remain in the studyfor Group 4.

All randomizations procedures were conducted by a biostatistician.

Analysis

The statistical analyses and data summaries were conducted by Dr. rer.hort. Martin Vanselow, Biometrie & Statistik, Zum Siemenshop 21, 30539Hannover, Germany, +49(0) 511 606 777 650, m.vanselow@t-online.de.

The main objective of the statistical analysis was the comparison of twoPRRS 94881 MLV vaccine groups (Groups 2 and 3) to an unvaccinatedchallenged control group (Group 1). All data were imported into SAS formanagement and evaluation. The data were received from the study sponsorin the form of verified SAS data sets. Cases which had been withdrawnfrom the study were considered for the respective parameter of analysisuntil date of exclusion. All data were summarized descriptively (m,minimum, maximum, mean, median, standard deviation, interquartile range,or frequency distributions, confidence interval) based on the type ofthe variable. The statistical analyses were performed using SAS softwarerelease 8.2 (SAS, 2001, Cary, N.C., USA: SAS Institute Inc.).

Variables for the Statistical Evaluation of the Study:

Primary Variables

Proportions of live piglets at farrowing/abortion (DOF+0)

Proportions of live piglets at 20 days of age (DOF+20)

Supportive Variables

Gilt clinical assessments post-vaccination

Gilt PRRS serology

Gilt viremia

Gilt clinical observations

Piglet viremia

Reproductive performance

Piglet clinical observations

Piglet average daily weight gain (ADWG)

Hypothesis to be Tested and Assumptions Made:

The unchallenged negative control group (Group 4) was excluded fromstatistical tests. The low titer and high titer groups (Groups 2 and 3,respectively) were compared to the challenge control group (Group 1).All tests between groups were designed as two-sided tests ondifferences. In the case of all tests, differences were considered asstatistically significant only if P≦0.05. Efficacy was demonstrated ifthe percentage or number of live born piglets and the percentage ornumber of weaned piglets at DOF+20 were significantly higher for one orboth vaccine groups compared with the challenged control group.

Details on Data Manipulations and Evaluation:

Clinical Assessments of Gilts Post-Vaccination

Frequency tables of animals with at least one positive finding betweenstudy days 1 and 21 and between study days 1 and 113 were generated.Differences between the challenge control and vaccine groups were testedby Fisher's exact test.

Clinical Observations of Gilts Post-Challenge

Frequency tables of animals with at least one positive finding betweenstudy day 116 and DOF+20 were generated. Differences between thechallenge control group and vaccine groups were tested by Fisher's exacttest.

Serology of Gilts

Frequency tables of “positive” ELISA results on study days 7, 14, 21,56, 84, 118, 125, 132 (pre-farrowing) and DOF+0, DOF+7, DOF+13 andDOF+20 were generated. Differences between the challenge control andvaccine groups were tested by Fisher's exact test.

Viremia of Gilts

Viremia data were evaluated for each day of investigation separately(pre-farrowing study days 7, 14, 21, 56, 84, 118, 125, 132 and DOF+0,DOF+7, DOF+13 and DOF+20). For the qualitative evaluation of the qPCRdata the analytical results ‘not detected’ (‘n.d.’) and ‘negative’ wereclassified as ‘negative’ and the analytical results ‘positive’ and ameasured value were classified as ‘positive’. For the quantitativeevaluation ‘not detected’ (‘n.d.’) and ‘negative’ were replaced by alog₁₀ GE/mL value of 0.0 and ‘positive’ was replaced by 3.0. Thequantitative PCR data (PRRS viral load [log₁₀ GE/mL]) were used forcomparisons between challenge control (group 1) and treatment groups 2and 3 using the Wilcoxon Mann-Whitney test. Frequency tables of‘positive’ qPCR results were generated. Differences between thechallenge control group and vaccine groups were tested by Fisher's exacttest.

Reproductive Performance

Absolute frequencies per gilt of total number, alive, healthy, weak,stillborn, dead and alive piglets at DOF+20 were determined and used assingle values for the comparisons between groups. Relative frequenciesper gilt of alive, healthy, weak, stillborn and dead piglets werecalculated in relation to the total number of piglets at farrowing andused as single values for the comparisons between groups. The percentageof live piglets per litter at DOF+20 was calculated in relation to thenumber of live piglets at farrowing minus number of mortalities andcrushed piglets. Differences between the challenge control group andvaccine groups were tested by the Wilcoxon Mann-Whitney test.

Viremia of Piglets

Viremia data were evaluated for each day of investigation separately(DOF+0, DOF+7, DOF+13 and DOF+20). For the qualitative evaluation of theqPCR data the analytical results ‘not detected’ (‘n.d.’) and ‘negative’were classified as ‘negative’ and the analytical results ‘positive’ anda measured value were classified as ‘positive’. The percentages of‘positive’ piglets per litter were calculated and used as single valuesfor the comparisons between groups by the Wilcoxon Mann-Whitney test.For the quantitative evaluation ‘not detected’ (‘n.d.’) and ‘negative’were replaced by a log₁₀ GE/mL value of 0.0 and ‘positive’ was replacedby 3.0. The median qPCR values per litter were calculated and used assingle values for the comparisons between groups by the WilcoxonMann-Whitney test. For the summary statistics the individual qPCR datawere used. The viral loads in lung samples were evaluated descriptivelyonly.

Body Weight and Average Daily Weight Gain of Piglets

Individual average daily weight gains (ADWG) were calculated for thetime periods between DOF+0 and DOF+20. Differences between treatmentgroups were tested by analysis of variance (ANOVA) and subsequentt-tests. Least squares means of the groups and differences between leastsquares means with 95% confidence intervals were derived from the ANOVA.The analysis for DOF+20 and ADWG was repeated with weight at DOF+0 as acovariate. The weight data of piglets per gilt were summarizeddescriptively.

Clinical Observations of Piglets

The percentage of piglets per litter with at least one positive findingbetween study days DOF+1 and DOF+20 were calculated and used as singlevalues for the comparisons between groups by the Wilcoxon Mann-Whitneytest. Data were analyzed assuming a completely random design structure.

Results

Gilt Reproductive Performance

Mean percentages of live piglets per litter at farrowing(healthy+weak+crushed/mortality) were 54.4%, 75.1%, 72.3%, and 93.0% forthe challenge control, low titer, high titer, and negative controlgroups, respectively. The low titer and high titer groups hadsignificantly higher percentage of live piglets per litter at farrowingcompared to the challenge control group (P≦0.0455). Mean number of livepiglets per litter at farrowing were 6.5, 8.3, 8.6 and 10.8 for thechallenge control, low titer, high titer, and negative control groups,respectively. No significant differences were detected between groupsfor the number of live piglets per litter at farrowing (P≧0.1039).

Mean percentages of healthy live piglets per litter were 41.4%, 65.8%,67.9%, and 93.0% for the challenge control, low titer, high titer, andnegative control groups, respectively. The low titer and high titergroups had significantly higher percentages of healthy live piglets perlitter at farrowing compared to the challenge control group (P≦0.0138).Mean number of healthy live piglets per litter at farrowing were 4.9,7.2, 8.1 and 10.8 for the challenge control, low titer, high titer, andnegative control groups, respectively. The high titer group had asignificantly higher number of healthy live piglets per litter atfarrowing (P=0.0211), while no difference was detected for the low titergroup in comparison with the challenge control group (P=0.0640).

Mean percentages of weak live piglets per litter at farrowing were 7.4%,7.1%, 0.4%, and 0.0% for the challenge control, low titer, high titer,and negative control groups, respectively. Mean number of weak livepiglets per litter at farrowing were 0.9, 0.8, 0.1 and 0.0 for thechallenge control, low titer, high titer, and negative control groups,respectively. The high titer group had significantly lower percentageand number of weak live piglets per litter at farrowing compared to thechallenge control group (P≦0.0090). Conversely, no differences weredetected between the low titer group and the challenge control group forpercentage or number of weak live piglets at farrowing (P≧0.8569).

Mean percentages of mummies per litter at farrowing were 28.1%, 14.1%,8.7%, and 0.0% for the challenge control, low titer, high titer, andnegative control groups, respectively. Mean number of mummies per litterat farrowing were 3.1, 1.6, 0.9, and 0.0 for the challenge control, lowtiter, high titer, and negative control groups, respectively. Both thelow titer and high titer groups had significantly lower percentages andnumbers of mummies per litter at farrowing compared with the challengecontrol group (P≦0.0190).

No significant differences were detected between the two vaccine titergroups and the challenge control group for percentage or number ofstillborn or mortalities/crushed piglets per litter at farrowing(P≧0.1681).

A summary of group reproductive performance results (% piglets perlitter and number of piglets per litter) on the DOF is shown below inTables 6.12 and 6.13.

TABLE 6.12 Summary of Group Reproductive Performance Results (% pigletsper litter) on the DOF Piglets Group * N Min. Max. Median Mean 95% CI SDP Alive 1 16 0 92 57.3 54.4 41.1 67.7 24.91 2 16 33 100 81.9 75.1 64.585.7 19.88 0.0184 3 16 17 100 75.6 72.3 59.5 85.1 24.01 0.0455 4 5 83100 91.7 93.0 84.2 101.8 7.11 Healthy 1 16 0 92 48.1 41.4 27.5 55.326.13 2 16 8 92 71.4 65.8 52.2 79.5 25.57 0.0138 3 16 17 100 71.8 67.954.4 81.3 25.25 0.0082 4 5 83 100 91.7 93.0 84.2 101.8 7.11 Weak 1 16 025 3.6 7.4 2.6 12.2 9.04 2 16 0 25 0.0 7.1 2.1 12.2 9.43 0.9441 3 16 0 70.0 0.4 −0.5 1.3 1.67 0.0024 4 5 0 0 0.0 0.0 0.0 0.0 0.00 Stillborn 1 160 50 9.5 17.5 8.5 26.4 16.83 2 16 0 42 3.8 10.7 3.3 18.2 13.94 0.1965 316 0 83 10.6 19.0 7.0 31.0 22.54 0.9033 4 5 0 17 8.3 7.0 −1.8 15.8 7.11Mummies 1 16 0 63 25.8 28.1 18.8 37.4 17.50 2 16 0 55 9.1 14.1 6.0 22.315.25 0.0190 3 16 0 50 3.3 8.7 1.2 16.3 14.14 0.0006 4 5 0 0 0.0 0.0 0.00.0 0.00 Mortalities/ 1 16 0 27 0.0 5.6 1.1 10.2 8.55 Crushed 2 16 0 180.0 2.1 −0.6 4.8 5.07 0.2276 3 16 0 25 0.0 4.0 0.1 7.8 7.25 0.6108 4 5 00 0.0 0.0 0.0 0.0 0.00 Alive on 1 15 0 100 33.3 43.6 23.0 64.3 37.28DOF + 20 2 16 13 100 84.5 73.8 58.5 89.2 28.80 0.0203 3 16 44 100 86.683.8 75.1 92.5 16.37 0.0022 4 5 100 100 100.0 100.0 100.0 100.0 0.00 *Group 1 = Challenge control group; Group 2 = Low titer PRRS 94881 MLVgroup; Group 3 = High titer PRRS 94881 MLV group; Group 4 = Negativecontrol group

TABLE 6.13 Summary of Group Reproductive Performance Results (No. ofpiglets per litter) on the DOF Piglets Group * N Min. Max. Median Mean95% CI SD P Total 1 16 6 15 12.0 11.6 10.4 12.9 2.31 2 16 9 13 11.0 11.110.4 11.7 1.24 0.1857 3 16 7 15 12.0 11.6 10.3 12.9 2.42 0.9623 4 5 1014 12.0 11.6 9.5 13.7 1.67 Alive 1 16 0 11 6.0 6.5 4.7 8.3 3.35 2 16 412 8.0 8.3 7.0 9.5 2.27 0.1543 3 16 2 13 9.0 8.6 6.6 10.6 3.77 0.1039 45 9 14 10.0 10.8 8.4 13.2 1.92 Healthy 1 16 0 11 5.5 4.9 3.1 6.7 3.36 216 1 12 7.0 7.2 5.7 8.7 2.83 0.0640 3 16 2 13 8.5 8.1 6.1 10.1 3.760.0211 4 5 9 14 10.0 10.8 8.4 13.2 1.92 Weak 1 16 0 3 0.5 0.9 0.3 1.51.09 2 16 0 3 0.0 0.8 0.2 1.4 1.11 0.8569 3 16 0 1 0.0 0.1 −0.1 0.2 0.250.0090 4 5 0 0 0.0 0.0 0.0 0.0 0.00 Stillborn 1 16 0 6 1.0 2.0 0.9 3.12.03 2 16 0 5 0.5 1.3 0.4 2.1 1.65 0.1681 3 16 0 10 1.0 2.1 0.8 3.5 2.580.9478 4 5 0 2 1.0 0.8 −0.2 1.8 0.84 Mummies 1 16 0 7 3.0 3.1 2.1 4.11.89 2 16 0 6 1.0 1.6 0.7 2.5 1.67 0.0125 3 16 0 4 0.5 0.9 0.2 1.5 1.200.0003 4 5 0 0 0.0 0.0 0.0 0.0 0.00 Mortalities/ 1 16 0 3 0.0 0.7 0.11.2 1.01 Crushed 2 16 0 2 0.0 0.3 −0.1 0.6 0.58 0.2200 3 16 0 2 0.0 0.40.0 0.8 0.73 0.6115 4 5 0 0 0.0 0.0 0.0 0.0 0.00 Alive on 1 16 0 10 2.02.9 1.2 4.7 3.21 DOF + 20 2 16 1 10 6.5 6.2 4.5 7.9 3.19 0.0063 3 16 212 7.5 6.9 5.0 8.9 3.71 0.0026 4 5 9 14 10.0 10.8 8.4 13.2 1.92 * Group1 = Challenge control group; Group 2 = Low titer PRRS 94881 MLV group;Group 3 = High titer PRRS 94881 MLV group; Group 4 = Negative controlgroup

Live Piglets at DOF+20

These scores highlight the number of live piglets at weaning (20 days ofage). A summary of group percentage and number of live piglets perlitter on DOF+20 is shown above in Tables 6.12 and 6.13.

Mean percentages of live piglets per litter at weaning (DOF+20) were43.6%, 73.8%, 83.8%, and 100.0% for the challenge control, low titer,high titer, and negative control groups, respectively. Mean number oflive piglets per litter at weaning were 2.9, 6.2, 6.9 and 10.8 for thechallenge control, low titer, high titer, and negative control groups,respectively. Both the low titer and high titer groups had significantlyhigher percentages and numbers of alive piglets per litter at weaning(DOF+20) compared with the challenge control group (P≦0.0203).

Gilt qPCR Viremia

All gilts were qPCR negative for PRRSv RNA on D0. All challenge controland negative control gilts remained qPCR negative for PRRSv RNA up toand including the day of challenge (D118). The negative control groupremained qPCR negative for the remainder of the study, with exception ofgilt No. 108, which was “positive” on DOF+7. Gilt No. 108 was negativeat other time points for PRRSv RNA by qPCR testing.

Post-vaccination, 50% and 36% of low titer and high titer gilts,respectively, were qPCR positive for PRRSv RNA (P≦0.0007) on D7. FromD14 to D56, only 4% of low titer gilts remained qPCR positive while upto 4% of high titer gilts were qPCR positive intermittently during thisobservation period. No differences were detected between vaccine groupsand the challenge control group from D14 to D56 for the percentage ofgilts qPCR positive for PRRSv RNA (P=1.0000 or no test conducted). Allvaccinated gilts were qPCR negative for PRRSv RNA on D84 and D118 (dayof challenge).

Post-challenge, the low titer and higher groups had statistically lowerpercentages of gilts qPCR positive for PRRSv RNA compared with thechallenge control group on Days 125, DOF 0, and DOF+13 (P≦0.0155). OnD132, the low titer group had a significantly lower percentage of giltsqPCR positive for PRRSv RNA (P=0.0290); while no statistical differencewas detected between the high titer group and the challenge controlgroup (P=0.1556). No significant differences were detected betweenvaccine groups and the challenge control group on DOF+7 and DOF+20 forthe percentage of gilts qPCR positive for PRRSv RNA (P≧0.1719).

A summary of group percentage of gilts qPCR positive for PRRSv RNA fromD7 to DOF+20 is shown below in Tables 6.14 and 6.15.

TABLE 6.14 Summary of Group Percentage of Gilts qPCR Positive for PRRSvRNA from D 7 to D 132 Study Day Group* N % 95% CI Total P 7 1 0 0 0.012.3 28 2 14 50 30.6 69.4 28 <0.0001  3 10 36 18.6 55.9 28 0.0007 4 0 00.0 30.8 10 14 1 0 0 0.0 12.3 28 2 1 4 0.1 18.3 28 1.0000 3 0 0 0.0 12.328 n.a. 4 0 0 0.0 30.8 10 21 1 0 0 0.0 12.3 28 2 1 4 0.1 18.3 28 1.00003 1 4 0.1 18.3 28 1.0000 4 0 0 0.0 30.8 10 56 1 0 0 0.0 14.8 23 2 1 40.1 19.6 26 1.0000 3 1 4 0.1 20.4 25 1.0000 4 0 0 0.0 33.6 9 84 1 0 00.0 14.8 23 2 0 0 0.0 13.2 26 n.a. 3 0 0 0.0 13.7 25 n.a. 4 0 0 0.0 33.69 1 0 0 0.0 20.6 16 118 (Day of 2 0 0 0.0 20.6 16 n.a. challenge) 3 0 00.0 20.6 16 n.a. 4 0 0 0.0 52.2 5 125 1 16 100 79.4 100.0 16 2 5 31 11.058.7 16 0.0001 3 4 25 7.3 52.4 16 <0.0001  4 0 0 0.0 52.2 5 132 1 10 6335.4 84.8 16 2 3 19 4.0 45.6 16 0.0290 3 5 31 11.0 58.7 16 0.1556 4 0 00.0 52.2 5 *Group 1 = Challenge control group; Group 2 = Low titer PRRS94881 MLV group; Group 3 = High titer PRRS94881 MLV group; Group 4 =Negative control group; n.a. = not applicable, no test conducted

TABLE 6.15 Summary of Group Percentage of Gilts qPCR Positive for PRRSvRNA from DOF 0 to DOF + 20 Study Day Group* N % 95% CI Total P DOF + 0 115 94 69.8 99.8 16 2 4 25 7.3 52.4 16 0.0002 3 1 6 0.2 30.2 16 <0.0001 40 0 0.0 52.2 5 DOF + 7 1 5 31 11.0 58.7 16 2 2 13 1.6 38.3 16 0.3944 3 16 0.2 30.2 16 0.1719 4 1 20 0.5 71.6 5 DOF + 13 1 7 47 21.3 73.4 15 2 00 0.0 20.6 16 0.0024 3 1 6 0.2 30.2 16 0.0155 4 0 0 0.0 52.2 5 DOF + 201 3 19 4.0 45.6 16 2 3 19 4.0 45.6 16 1.0000 3 0 0 0.0 20.6 16 0.2258 40 0 0.0 52.2 5 *Group 1 = Challenge control group; Group 2 = Low titerPRRS 94881 MLV group; Group 3 = High titer PRRS 94881 MLV group; Group 4= Negative control group

Both vaccine groups had significantly higher median viral loads comparedwith the challenge control group on D7 (P≦0.0007). No differences weredetected between vaccine groups and the challenge control group from D14to D56 for viral load (P=1.0000). All vaccinated gilts had zero viralload on D84 and D118 (day of challenge).

Post-challenge, the low titer and higher groups had statistically lowermedian viral loads compared with the challenge control group on D125,DOF 0, and DOF+13 (P≦0.0155). On D132, the low titer group had asignificantly lower median viral load (P=0.0230); while no statisticaldifference was detected between the high titer group and the challengecontrol group (0.94 and 1.97 log₁₀ GE/mL respectively; P=0.1144). Nosignificant differences for viral load were detected between vaccinegroups and the challenge control group on DOF+7 and DOF+20 (P≧0.1719).

A summary of group mean gilt qPCR GE/mL results from D7 to DOF+20 isshown below in Tables 6.16 and 6.17.

TABLE 6.16 Summary of Group Gilt qPCR Results (log₁₀ GE/mL) From D 7 toD 132 Study Day Group * N Min. Max. Median 95% CI QRange Mean P 7 1 280.00 0.00 0.000 0.000 0.000 0.000 0.000 2 28 0.00 3.00 1.500 0.000 3.0003.000 1.500 <0.0001 3 28 0.00 3.00 0.000 0.000 3.000 3.000 1.071 0.00074 10 0.00 0.00 0.000 0.000 0.000 0.000 0.000 14 1 28 0.00 0.00 0.0000.000 0.000 0.000 0.000 2 28 0.00 3.00 0.000 0.000 0.000 0.000 0.1071.0000 3 28 0.00 0.00 0.000 0.000 0.000 0.000 0.000 1.0000 4 10 0.000.00 0.000 0.000 0.000 0.000 0.000 21 1 28 0.00 0.00 0.000 0.000 0.0000.000 0.000 2 28 0.00 3.00 0.000 0.000 0.000 0.000 0.107 1.0000 3 280.00 3.00 0.000 0.000 0.000 0.000 0.107 1.0000 4 10 0.00 0.00 0.0000.000 0.000 0.000 0.000 56 1 23 0.00 0.00 0.000 0.000 0.000 0.000 0.0002 26 0.00 3.00 0.000 0.000 0.000 0.000 0.115 1.0000 3 25 0.00 3.00 0.0000.000 0.000 0.000 0.120 1.0000 4 9 0.00 0.00 0.000 0.000 0.000 0.0000.000 84 1 23 0.00 0.00 0.000 0.000 0.000 0.000 0.000 2 26 0.00 0.000.000 0.000 0.000 0.000 0.000 1.0000 3 25 0.00 0.00 0.000 0.000 0.0000.000 0.000 1.0000 4 9 0.00 0.00 0.000 0.000 0.000 0.000 0.000 118 (Day1 16 0.00 0.00 0.000 0.000 0.000 0.000 0.000 of 2 16 0.00 0.00 0.0000.000 0.000 0.000 0.000 1.0000 challenge) 3 16 0.00 0.00 0.000 0.0000.000 0.000 0.000 1.0000 4 5 0.00 0.00 0.000 0.000 0.000 0.000 0.000 1251 16 3.00 5.38 4.495 4.130 4.880 0.765 4.419 2 16 0.00 6.46 0.000 0.0003.000 3.000 1.293 0.0001 3 16 0.00 3.00 0.000 0.000 3.000 1.500 0.750<0.0001 4 5 0.00 0.00 0.000 0.000 0.000 0.000 0.000 132 1 16 0.00 4.473.000 0.000 3.000 3.000 1.967 2 16 0.00 3.00 0.000 0.000 0.000 0.0000.563 0.0230 3 16 0.00 3.00 0.000 0.000 3.000 3.000 0.938 0.1144 4 50.00 0.00 0.000 0.000 0.000 0.000 0.000 * Group 1 = Challenge controlgroup; Group 2 = Low titer PRRS 94881 MLV group; Group 3 = High titerPRRS 94881 MLV group; Group 4 = Negative control group

TABLE 6.17 Summary of Group Gilt qPCR Results (log₁₀ GE/mL) From DOF 0to DOF + 20 Study Day Group * N Min. Max. Median 95% CI QRange Mean PDOF + 0 1 16 0.00 3.00 3.000 3.000 3.000 0.000 2.813 2 16 0.00 3.000.000 0.000 3.000 1.500 0.750 0.0002 3 16 0.00 3.00 0.000 0.000 0.0000.000 0.188 <0.0001 4 5 0.00 0.00 0.000 0.000 0.000 0.000 0.000 DOF + 71 16 0.00 3.00 0.000 0.000 3.000 3.000 0.938 2 16 0.00 5.55 0.000 0.0000.000 0.000 0.534 0.3944 3 16 0.00 3.00 0.000 0.000 0.000 0.000 0.1880.1719 4 5 0.00 3.00 0.000 0.000 3.000 0.000 0.600 DOF + 13 1 15 0.003.00 0.000 0.000 3.000 3.000 1.400 2 16 0.00 0.00 0.000 0.000 0.0000.000 0.000 0.0024 3 16 0.00 3.00 0.000 0.000 0.000 0.000 0.188 0.0155 45 0.00 0.00 0.000 0.000 0.000 0.000 0.000 DOF + 20 1 16 0.00 3.00 0.0000.000 0.000 0.000 0.563 2 16 0.00 6.45 0.000 0.000 0.000 0.000 0.9030.7924 3 16 0.00 0.00 0.000 0.000 0.000 0.000 0.000 0.2258 4 5 0.00 0.000.000 0.000 0.000 0.000 0.000 * Group 1 = Challenge control group; Group2 = Low titer PRRS 94881 MLV group; Group 3 = High titer PRRS 94881 MLVgroup; Group 4 = Negative control group

Gilt Clinical Observations Scores Post-Challenge

From D116 to DOF+20, 25%, 25%, 38% and 60% of challenge control, lowtiter, high titer and negative control gilts, respectively, exhibitedclinical disease for at least one day from D116 to DOF+20. Nosignificant differences were detected between vaccine groups and thechallenge control group for the frequency of gilts positive for clinicaldisease from D116 to DOF+20 (P≧0.7043).

A summary of group percentage of gilts positive for clinical disease (aclinical observation score of >0) for at least one day from D116 toDOF+20 is shown below in Table 6.18.

TABLE 6.18 Summary of group percentage of gilts positive for clinicaldisease (a clinical observation score of > 0) for at least one day fromD 116 to DOF + 20 Study Days Group* N % 95% CI Total P 116- 1 4 25 7.352.4 16 DOF + 20 2 4 25 7.3 52.4 16 1.0000 3 6 38 15.2 64.6 16 0.7043 43 60 14.7 94.7 5 *Group 1 = Challenge control group; Group 2 = Low titerPRRS 94881 MLV group; Group 3 = High titer PRRS 94881 MLV group; Group 4= Negative control group

Gilt PRRS ELISA Serology

All gilts were PRRS seronegative on D0 and D7. All challenge control andnegative control gilts remained PRRS seronegative up to and includingthe day of challenge (D118); while the negative control group remainedPRRS seronegative for the remainder of the study (DOF+20).

On D14, 18% and 21% of low titer and high titer gilts, respectively,were PRRS seropositive. The high titer group had a significantly higherpercentage of PRRS seropositive gilts on D14 (P=0.0232), while nodifference was detected for the low titer group in comparison with thechallenge control group (p=0.0515). These percentages reached grouphighs of 65% and 60% for the low titer and high titer groups,respectively on D56 (P<0.0001). On the day of the challenge (D118), 56%and 50% of low titer and high titer gilts were PRRS seropositive(P≦0.0024). On D125, 6%, 88%, and 100% of challenge control, low titerand high titer gilts, respectively, were PRRS seropositive; and thedifference between the vaccine groups and the challenge control groupwere significant (P<0.0001). After D125, all remaining challengecontrol, low titer and high titer gilts were PRRS seropositive for theremainder of the study (no test conducted).

A summary of group PRRS ELISA serology results from D14 to DOF+20 isshown below in Tables 6.19 and 6.20.

TABLE 6.19 Summary of Group Gilt PRRS ELISA Serology Results from D 14to Day 132 Study Day Group* N % 95% CI Total P 7 1 0 0 0.0 12.3 28 2 0 00.0 12.3 28 n.a. 3 0 0 0.0 12.3 28 n.a. 4 0 0 0.0 30.8 10 14 1 0 0 0.012.3 28 2 5 18 6.1 36.9 28 0.0515 3 6 21 8.3 41.0 28 0.0232 4 0 0 0.030.8 10 21 1 0 0 0.0 12.3 28 2 13 46 27.5 66.1 28 <0.0001 3 11 39 21.559.4 28 0.0003 4 0 0 0.0 30.8 10 56 1 0 0 0.0 14.8 23 2 17 65 44.3 82.826 <0.0001 3 15 60 38.7 78.9 25 <0.0001 4 0 0 0.0 33.6 9 84 1 0 0 0.014.8 23 2 15 58 36.9 76.6 26 <0.0001 3 14 56 34.9 75.6 25 <0.0001 4 0 00.0 33.6 9 118 1 0 0 0.0 20.6 16 2 9 56 29.9 80.2 16 0.0008 3 8 50 24.775.3 16 0.0024 4 0 0 0.0 52.2 5 125 1 1 6 0.2 30.2 16 2 14 88 61.7 98.416 <0.0001 3 16 100 79.4 100.0 16 <0.0001 4 0 0 0.0 52.2 5 132 1 16 10079.4 100.0 16 2 16 100 79.4 100.0 16 n.a. 3 16 100 79.4 100.0 16 n.a. 40 0 0.0 52.2 5 *Group 1 = Challenge control group; Group 2 = Low titerPRRS 94881 MLV group; Group 3 = High titer PRRS 94881 MLV group; Group 4= Negative control group, n.a. = not applicable, no test conducted

TABLE 6.20 Summary of Group Gilt PRRS ELISA Serology Results from DOF 0to DOF + 20 Study Day Group* N % 95% CI Total P DOF + 0 1 16 100 79.4100.0 16 2 16 100 79.4 100.0 16 n.a. 3 16 100 79.4 100.0 16 n.a. 4 0 00.0 52.2 5 DOF + 7 1 15 100 78.2 100.0 15 2 16 100 79.4 100.0 16 n.a. 316 100 79.4 100.0 16 n.a. 4 0 0 0.0 52.2 5 DOF + 13 1 16 100 79.4 100.016 2 16 100 79.4 100.0 16 n.a. 3 16 100 79.4 100.0 16 n.a. 4 0 0 0.052.2 5 DOF + 20 1 16 100 79.4 100.0 16 2 16 100 79.4 100.0 16 n.a. 3 15100 78.2 100.0 15 n.a. 4 0 0 0.0 52.2 5 *Group 1 = Challenge controlgroup; Group 2 = Low titer PRRS 94881 MLV group; Group 3 = High titerPRRS 94881 MLV group; Group 4 = Negative control group. **No testconducted on sample from Gilt No. 106. n.a. = not applicable, no testconducted

Gilt Assessments Post-Vaccination

No abnormal assessments from D1 to 21 were detected in any groups and notest was conducted. From D1 to D113, 4%, 4%, 0% and 10% of challengecontrol, low titer, high titer and negative control gilts, respectively,exhibited an abnormal assessment for at least one day from D1 to D113.No significant differences were detected between vaccine groups and thechallenge control group for abnormal assessments from D1 to D113(P=1.0000).

Individually, No. 109 (challenge control group) exhibited lameness ofthe right rear leg on D85, No. 12 (low titer group) exhibited a swellingin the left neck region from D78 to D89, and No. 21 (negative controlgroup) exhibited lameness from D81 to D83.

A summary of group percentage of gilts that exhibited an abnormalassessment for at least one day from D1 to D21 and from D1 to D113 isshown below in Table 6.21.

TABLE 6.21 Summary of Group Abnormal Assessments for At Least One Dayfrom D 1 to D 113 Study Days Group* N % 95% CI Total P 1-21  1-4 no‘positive’ findings 1-113 1 1 4 0.1 18.3 28 2 1 4 0.1 18.3 28 1.0000 3 00 0.0 12.3 28 1.0000 4 1 10 0.3 44.5 10 *Group 1 = Challenge controlgroup; Group 2 = Low titer PRRS 94881 MLV group; Group 3 = High titerPRRS 94881 MLV group; Group 4 = Negative control group

Piglet Clinical Observations Scores

Mean percentages of piglets per litter positive for clinical disease (aclinical observation score of >0) for at least one day from DOF+1 toDOF+20 were 91.6%, 32.5%, 33.4% and 3.2% for the challenge control, lowtiter, high titer, and negative control groups, respectively. Low andhigh titer groups had significantly lower percentages of piglets perlitter positive for clinical disease for at least one day from DOF+1 toDOF+20 compared with the challenge control group (p≦0.0001).

A summary of group percentage of piglets per litter that were positivefor clinical disease (a clinical observation score of >0) for at leastone day from DOF+1 to DOF+20 is shown below in Table 6.22.

TABLE 6.22 Summary of group percentage of piglets per litter positivefor clinical disease (a clinical observation score of > 0) for at leastone day from DOF + 1 to DOF + 20 Study Days Group* N Min. Max. MedianMean 95% CI SD P DOF + 1 1 15 56 100 100.0 91.6 82.9 100.4 15.78 to 2 160 100 25.0 32.5 15.6 49.4 31.64 <0.0001 DOF + 20 3 16 0 100 25.0 33.419.0 47.9 27.16 <0.0001 4 5 0 9 0.0 3.2 −2.3 8.8 4.50 *Group 1 =Challenge control group; Group 2 = Low titer PRRS 94881 MLV group; Group3 = High titer PRRS 94881 MLV group; Group 4 = Negative control group

Piglet Serum/Body Fluids qPCR Results

A mean of 86.3%, 58.1%, 55.0% and 0% of piglets per litter for thechallenge control, low titer, high titer and negative control groups,respectively, were qPCR positive for PRRSv RNA on DOF 0. The low titerand higher groups had statistically lower percentages of piglets perlitter qPCR positive for PRRSv RNA compared with the challenge controlgroup on DOF 0 (P≦0.0381). On DOF+7, again the low titer and high titergroups had significantly lower percentages of piglets per litter qPCRpositive for PRRSv RNA compared with the challenge control group(P≦0.0293). On DOF+13, only the low titer group had a significantlylower percentage of piglets per litter qPCR positive (P=0.0216); whileno significant differences were detected for the high titer group andthe challenge control for the percentage of piglets per litter qPCRpositive (P=0.0860). No significant differences were detected betweengroups on DOF+20 (P≧0.0614).

A summary of group percentage of serum/body fluid qPCR PRRSv positivepiglets per gilt is shown below in Table 6.23.

TABLE 6.23 A Summary of Group Percentage of Serum/Body Fluid qPCR PRRSvPositive Piglets per Gilt Study Day Group* N Min. Max. Median Mean 95%CI SD P DOF + 0 1 16 50 100 96.4 86.3 76.8 95.8 17.87 2 16 0 100 68.358.1 37.3 78.9 39.07 0.0381 3 16 0 100 60.0 55.0 37.0 73.0 33.77 0.00184 5 0 0 0.0 0.0 0.0 0.0 0.00 DOF + 7 1 12 100 100 100.0 100.0 100.0100.0 0.00 2 16 10 100 100.0 76.6 57.1 96.0 36.51 0.0293 3 16 0 100100.0 78.6 60.6 96.6 33.83 0.0175 4 5 0 0 0.0 0.0 0.0 0.0 0.00 DOF + 131 11 100 100 100.0 100.0 100.0 100.0 0.00 2 16 0 100 100.0 75.4 55.095.8 38.31 0.0216 3 16 0 100 100.0 84.0 68.2 99.9 29.75 0.0860 4 5 0 00.0 0.0 0.0 0.0 0.00 DOF + 20 1 11 0 100 100.0 90.9 70.7 111.2 30.15 216 0 100 93.8 75.3 55.6 95.0 36.97 0.0614 3 16 0 100 100.0 81.6 65.098.1 31.06 0.1832 4 5 0 0 0.0 0.0 0.0 0.0 0.00 *Group 1 = Challengecontrol group; Group 2 = Low titer PRRS 94881 MLV group; Group 3 = Hightiter PRRS 94881 MLV group; Group 4 = Negative control group

The high titer group had a significantly lower median qPCR resultcompared with the challenge control group on DOF 0 (P=0.0030); while nodifference was detected between the low titer group and the challengecontrol group (P=0.0620). On DOF+7, DOF+13 and DOF+20, both vaccinegroups had significantly lower median qPCR values compared with thechallenge control group (p≦0.0122).

A summary of group piglet serum/body fluid qPCR GE/mL results per giltis shown below in Table 6.24.

TABLE 6.24 Summary of Group Piglet Serum/Body Fluid qPCR results (log₁₀GE/mL) per Gilt (P values for differences between groups based on medianqPCR values) Study Day Group * N Min. Max. Median 95% CI QRange Mean PDOF + 0 1 180 0.00 8.69 6.400 6.080 6.790 3.195 5.556 2 176 0.00 8.473.000 3.000 4.420 6.945 3.560 0.0620 3 183 0.00 8.76 3.000 0.000 3.0006.580 3.049 0.0030 4 58 0.00 0.00 0.000 0.000 0.000 0.000 0.000 DOF + 71 58 4.47 8.76 6.950 6.610 7.370 1.300 6.914 2 103 0.00 8.12 3.000 3.0004.930 5.640 3.337 <0.0001 3 115 0.00 6.91 4.280 3.000 4.630 2.120 3.642<0.0001 4 54 0.00 0.00 0.000 0.000 0.000 0.000 0.000 DOF + 13 1 52 4.198.62 6.835 6.430 6.970 0.995 6.549 2 100 0.00 8.22 3.000 3.000 3.0004.530 2.678 <0.0001 3 113 0.00 6.54 3.000 3.000 3.000 1.580 3.413<0.0001 4 54 0.00 0.00 0.000 0.000 0.000 0.000 0.000 DOF + 20 1 46 0.006.94 5.595 5.270 6.520 1.770 5.554 2 98 0.00 6.59 3.000 3.000 3.0004.000 2.502 0.0122 3 111 0.00 6.28 3.000 3.000 3.000 1.160 3.218 0.00054 54 0.00 0.00 0.000 0.000 0.000 0.000 0.000 * Group 1 = Challengecontrol group; Group 2 = Low titer PRRS 94881 MLV group; Group 3 = Hightiter PRRS 94881 MLV group; Group 4 = Negative control group

Piglet ADWG

No differences were detected between groups for LS Mean body weights onDOF 0 (p≧0.2972). Both vaccine groups had higher least square mean bodyweights compared with the challenge control group on DOF+20 (P<0.0028),with or without DOF 0 body weights factored as a covariate in theanalyses.

Mean ADWG from DOF 0 to DOF+20 were 0.1 kg/day, 0.2 kg/day, 0.2 kg/dayand 0.2 kg/day for the challenge control, low titer, high titer, andnegative control groups, respectively. Both vaccine groups hadsignificantly higher ADWG compared with the challenge control group(P<0.0028), with or without DOF 0 body weights factored as a covariatein the analyses.

A summary of group DOF 0 and DOF+20 piglet body weights and DOF 0 toDOF+20 ADWG (kg/day) is shown below in Tables 6.25 and 6.26.

TABLE 6.25 Summary of Group DOF 0 and DOF + 20 Piglet Body Weights andDOF 0 to DOF + 20 ADWG (kg/day) Study Day(s) Group* N Min. Max. MedianMean 95% CI SD DOF + 0 1 47 0.9 2.0 1.40 1.34 1.274 1.411 0.234 Body 299 0.9 2.1 1.40 1.43 1.388 1.479 0.227 Weights 3 111 0.9 2.0 1.40 1.401.360 1.448 0.234 4 54 0.9 1.9 1.40 1.39 1.335 1.454 0.218 DOF + 20 1 471.5 6.1 3.70 3.80 3.462 4.146 1.164 Body 2 99 2.4 8.3 5.50 5.42 5.1685.673 1.266 Weights 3 111 2.1 8.2 5.30 5.19 5.000 5.388 1.032 4 54 2.46.9 5.20 5.26 5.008 5.511 0.922 ADWG 1 47 0.015 0.235 0.1150 0.12310.10649 0.13968 0.05653 (DOF + 0 to 2 99 0.065 0.340 0.2000 0.19930.18770 0.21099 0.05837 DOF + 3 111 0.055 0.330 0.1950 0.1895 0.180780.19823 0.04638 20) 4 54 0.060 0.260 0.1925 0.1932 0.18305 0.203430.03733 *Group 1 = Challenge control group; Group 2 = Low titer PRRS94881 MLV group; Group 3 = High titer PRRS 94881 MLV group; Group 4 =Negative control group

TABLE 6.26 Summary of Group LS Mean Body Weights and DOF 0 to DOF + 20ADWG (kg/day)—Test Results (P values) on Differences between GroupsStudy Day(s) Group* LS Mean 95% confidence interval P DOF + 0 1 1.321.169 1.477 Body 2 1.42 1.318 1.522 Weights 3 1.41 1.317 1.497 Diff. 1-2−0.10 −0.281 0.088 0.2972 Diff. 1-3 −0.08 −0.262 0.094 0.3467 DOF + 20 13.82 3.072 4.567 Body 2 5.32 4.827 5.819 Weights 3 5.35 4.910 5.785Diff. 1-2 −1.50 −2.401 −0.606 0.0016 Diff. 1-3 −1.53 −2.394 −0.6620.0010 D0F + 20** 1 4.01 3.341 4.685 Body 2 5.28 4.843 5.727 Weights 35.34 4.950 5.728 Diff. 1-2 −1.27 −2.078 −0.466 0.0028 Diff. 1-3 −1.33−2.103 −0.550 0.0013 ADWG 1 0.125 0.0903 0.1594 (DOF + 0 to 2 0.1950.1722 0.2181 DOF + 20) 3 0.197 0.1768 0.2172 Diff. 1-2 −0.070 −0.1118−0.0289 0.0014 Diff. 1-3 −0.072 −0.1122 −0.0322 0.0008 ADWG 1 0.1300.0969 0.1640 (DOF + 0 to 2 0.194 0.1720 0.2161 DOF + 20**) 3 0.1970.1773 0.2162 Diff. 1-2 −0.064 −0.1039 −0.0233 0.0028 Diff. 1-3 −0.066−0.1052 −0.0275 0.0013 *Group 1 = Challenge control group; Group 2 = Lowtiter PRRS 94881 MLV group; Group 3 = High titer PRRS 94881 MLV group;Group 4 = Negative control group. **weight at DOF + 0 was used as acovariate

Piglet Necropsy Observations and Diagnoses

Feti listed as stillborns, mummies or crushed at farrowing wereconfirmed at necropsy as correctly categorized with the exception of 8feti. Two-challenge control feti were listed as stillborns (40-S1,66-S1), but necropsy results revealed inflated lungs indicating theywere alive at the time of birth. Two-challenge control feti were listedas crushed (1-C1, 79-C2), but necropsy results revealed non-inflatedlungs for both feti, indicating they did not breath. One-low titer fetuswas listed as a stillborn (85-S2), but necropsy results revealedinflated lungs indicating the piglet was alive at the time of birth.Three-high titer piglets were listed as crushed (36-C1, 36-C2, 65-C1),but necropsy results revealed non-inflated lungs for both feti,indicating they did not breath. Due to the low number of fetiincorrectly listed at time of farrowing, no changes were made to giltperformance analyses.

One-challenge control piglet 102-428 died subsequently to bloodcollection, which was confirmed by necropsy.

Piglet Lung qPCR Results

Of the feti and dead piglets necropsied, the mean lung qPCR results were4.68, 4.09, 3.55 and 0.0 log₁₀ GE/mL for the challenge control, lowtiter, high titer, and negative control groups, respectively. Nostatistical analyses were conducted on these data.

A summary of group lung PRRSv qPCR results (log₁₀ GE/mL) is shown belowin Table 6.27.

TABLE 6.27 Summary of Group Piglet Lung PRRSv qPCR Results (log₁₀ GE/mL)Group N Min. Max. Median 95% CI QRange Mean 1 141 0.00 7.95 5.140 4.8105.390 2.990 4.676 2 79 0.00 7.45 4.780 3.000 5.260 2.620 4.092 3 75 0.006.84 4.220 3.000 5.100 5.620 3.547 4 4 0.00 0.00 0.000 0.000 0.000 0.0000.000 *Group 1 = Challenge control group; Group 2 = Low titer PRRS 94881MLV group; Group 3 = High titer PRRS 94881 MLV group; Group 4 = Negativecontrol group

Discussion/Conclusion

To achieve the study objective, four groups of PRRS susceptible giltswere included in the study design on D0: a challenge control group thatreceived control product (Group 1); a low titer vaccine group thatreceived 1×10^(2.43) TCID₅₀ of PRRS 94881 MLV (IVP No. 1, Group 2); ahigh titer vaccine group that received 1×10^(3.90) TCID₅₀ of PRRS 94881MLV (IVP No. 2, Group 3); and a negative control group (Group 4) thatalso received control product. Each treatment was administered as a 2.0mL dose IM at approximately 28 days prior to breeding (D0).

To determine the minimum immunizing dose of PRRS 94881 MLV, the twovaccine titer groups and the challenge control group were challenged onD118 (approximately 90 days of gestation) with a heterologous Europeanisolate of PRRSv (isolate 190136) and evaluated post-challenge forpercentage and number of live piglets per litter at birth (day offarrowing, DOF) and percentage and number of live piglets per litter at21 days of age (DOF+21).

Validation of the Study (Negative Control Group 4)

To ensure that source gilts were free of PRRSv and that no extraneousPRRSv exposure or cross-contamination among treatment and control groupsoccurred during the study, a negative control group (Group 4) wasincluded in the study design. Negative control gilts were negative forPRRS antibodies throughout the study. In addition, this group of giltsand their progeny were also negative for PRRSv viremia (qPCR) at alltested time points with exception of No. 108 on DOF+7. Gilt No. 108 was“positive” on DOF+7, while qPCR negative at all other time points andher piglets were negative for PRRSv RNA as well. This result wasconsidered an error due to sample contamination and not due to PRRSvinfection. These results support that the negative control groupremained free of PRRS infection during the study and validate theresults of this trial.

Validation of the PRRSv Reproductive Challenge Model (Challenge ControlGroup 1)

A challenge model involving a virulent EU-derived strain of PRRSv thatinduces sufficient and reproducible PRRS clinical disease is necessaryto adequately evaluate PRRS vaccine efficacy in a laboratory setting.Following inoculation with European PRRS isolate 190136 (1×10^(6.30)TCID₅₀/6 mL), the challenge control group exhibited only 54.4% livepiglets per litter at birth (93.0% for the negative control group),17.5% and 28.1% per litter of stillborns and mummies, respectively (7.0%and 0.0%, respectively, for the negative control group), 91.6% pigletsper litter exhibited clinical disease for at least one day from DOF+1 toDOF+20 (3.2% piglets per litter for the negative control group), a meanof 2.9 live piglets per litter at 20 days of age (a mean of 10.8 for thenegative control group), and 86.3% of piglets per litter viremic atbirth (0% for the negative control group). These results highlight thatsevere PRRS-specific clinical disease was induced in the unvaccinated,challenge control group of gilts and their progeny, thus validating thischallenge model as an adequate clinical laboratory tool to evaluate PRRSvaccine efficacy and more specifically, the MID of PRRS 94881 MLV ingilts.

Determination of the minimum immunizing dose of PRRS 94881 MLV in gilts(low and high titer vaccine doses; Groups 2-3)

Determination of the MID of PRRS 94881 MLV in gilts was based upon thevaccine group that received the lowest titer of vaccine that resulted inhigher percentages or number of live piglets per litter at birth andhigher percentages of number of live piglets per litter at 20 days ofage post-challenge compared with the challenge control group.

Live piglets per litter (either percent or number) at farrowing wasselected as one of two key criteria for determining the MID of PRRS94881 MLV. The first key criterion was based upon the fact that PRRSvinfection in pregnant gilts and sows typically results in stillborns andmummies, with low numbers of live piglets at farrowing. Live piglets perlitter at birth were defined as the summation of healthy live, weak liveand crushed-mortality piglets at farrowing. Piglets listed as crushed ormortality were included in the “live” category because necropsy findingsconfirmed these piglets were alive at birth and died shortly thereafterdue to trauma. Both the low titer and high titer groups exhibitedsignificantly higher percentages of live piglets per litter at farrowingcompared with the challenge control (P≦0.0455), thus this criterion forvaccine efficacy was met. Although no significant differences weredetected between the low and high titer vaccine groups and the challengecontrol group with respect to the mean number of live piglets per litterat farrowing (P≧0.1857), the low titer and high titer groups did exhibitconsiderably higher mean number of live piglets per litter at farrowing(mean 8.3 and 8.6 piglets per litter, respectively) in relationship tothe challenge control group (mean 6.5 piglets per litter) thus,providing further evidence and support that a beneficial vaccinetreatment effect was observed in these animals post-challenge.

Live piglets per litter (either percent or number) at 20 days of age wasthe second criterion for determining the MID of PRRS 94881 MLV becausegilt PRRS immunity will influence in utero infection of piglets andshedding of virus from gilts to live piglets. Piglets infected with PRRSin utero and born alive or infected with virulent PRRS post-farrowingvia shedding from the gilt usually die before weaning secondary to PRRS.In this study, the challenge control, low titer, high titer and negativecontrol group exhibited 43.6%, 73.8%, 83.8% and 100% live piglets perlitter, respectively, at 20 days of age (P≦0.0203). Likewise, thechallenge control, low titer, high titer and negative control groups hada mean number of 2.9, 6.2, 6.9 and 10.8 piglets per litter,respectively, at 20 days of age (P≦0.0063). Both vaccine groups hadsignificantly higher percentage and number of live piglets at weaning(P≦0.0203), thus this criterion of the study objective was met.

Further analyses of farrowing data revealed more information thatsupports vaccine efficacy following PRRSv challenge, especially withrespect to the high titer group. The high titer group exhibitedstatistically a higher percentage and a higher mean number of healthypiglets at birth (P≦0.0211); while exhibiting significantly lowerpercentages and mean numbers of weak and mummified feti (P≦0.0090), incomparison with the challenge control group. These data support that thehigh vaccine dose induced protective immunity against a virulent andheterologous PRRSv challenge strain. The low titer group also exhibitedvaccine efficacy at farrowing, as evident by a higher percentage ofhealthy live piglets per litter (P=0.0138) and significantly lowerpercentages and mean numbers of mummified feti (P≦0.0190). Conversely,no differences were detected between groups for the percentage or numberof stillborn feti or crushed/mortalities at farrowing (P≧0.1681).

Seven days after challenge (D125), the low titer and high titer groupshad significantly lower percentages of gilts positive for PRRSv RNA byqPCR testing, as well as significantly lower viral load for both groups,in comparison to the challenge control group (P≦0.0001). These datafurther support that both vaccine dose levels induced adequate immunityin gilts to significantly lower viral replication following challenge.Likewise, the low and high titer groups had significantly lowerpercentages of gilts qPCR positive on DOF 0 and DOF+13, as well as lowerviral load for both groups on these study days (P≦0.0155). The low titergroup had significantly lower percentage of gilts qPCR positive andlower viral load on D132 (P≦0.0290); while no statistical differenceswere detected between the high titer and the challenge control group forthe same set of parameters (P≧0.1144). No statistical differences weredetected between vaccine groups and challenge control group forpercentage of gilts qPCR positive or viral load on DOF+7 and DOF+20(P≧0.1719).

Typically PRRSv does not induce clinical disease in gilts and sows,other than abortion. In this study 25%, 25%, 38% and 60% of challengecontrol, low titer, high titer and negative control gilts, respectively,exhibited clinical disease (received a clinical observation score >0)for at least one day post-challenge. No significant differences weredetected between the vaccine groups and the challenge control group withrespect to percentage of gilts with clinical disease for at least oneday from D116 to DOF+20 (P≧0.7043). Gilts that exhibited some form ofclinical disease did so at peri-parturition and not immediately afterchallenge. The high percentage of negative control gilts (60%) thatexhibited clinical disease and the fact that clinical disease was notedprimarily around the time of farrowing for all groups in this studysupports that clinical disease was not attributed to PRRS disease butrather to physiological changes associated with parturition.

All gilts in the study were PRRS ELISA seronegative on D0 thus providingconfirmation of the inclusion criteria for the test animals entering thestudy. Likewise, all gilts were PRRS ELISA seronegative on D7.Vaccinated gilts began to exhibit PRRS ELISA seropositive results on D14and the low and high dose groups exhibited their highest rate ofseroconversion of 65% and 60%, respectively, on D56 (P<0.0001).Conversely, the challenge control group remained PRRS ELISA seronegativeuntil 7 days post-challenge (D125). From D132 to study conclusion, alllow titer, high titer and challenge control gilts were PRRS ELISAseropositive. The percentage of viremia positive gilts post-vaccinationpeaked on D7 for both vaccine groups as evidenced by 50% and 36% for thelow and high titer groups, respectively (P≦0.0007). Viremia quicklydropped to 4% (1 of 28, No. 64) and 0% (0 of 28) for the low titer andhigh titer groups, respectively on D14 (P=1.0000 or no test conducted).Viremia remained at 4% for low titer (1 of 28, No. 56) and high titer (1of 28, No. 91) groups on D21. On D56, one of 26 (4%, No. 89) low titergilts and one of 25 (4%, No. 66) high titer gilts were positive forviremia. All gilts were negative for viremia on D84 and D118.

No significant differences were detected between both vaccine titergroups and the challenge control group with respect to percentage ofgilts per group post-vaccination with an abnormal clinical assessmentfor at least one day from D1 to D113 (P=1.0000). Individually, onlythree gilts exhibited any abnormal assessments during this time frame.Two gilts exhibited lameness (one challenge control gilt and onenegative control gilt) and one—low titer gilt exhibited swelling in theleft neck region. Since vaccine was administered in the right neckregion, no adverse events associated with this vaccine were noted.

Piglet PRRS viremia results on the DOF gave further insight to the levelof protection in gilts in preventing cross-placental infection ofpiglets. On the DOF, a mean of 58.1% and 55.0% piglets per gilt in thelow titer and high titer groups, respectively, were qPCR positive.Conversely, a mean of 86.3% piglets per gilt in the challenge controlgroup were qPCR positive in serum/body fluids, which was significantlyhigher than both vaccine groups (P≦0.0381). When piglet viral load onDOF 0 was examined, high titer piglets had significantly lower viralload in comparison to challenge control piglets (P=0.0030); while nodifference was detected for viral load between low titer and challengecontrol piglets (P=0.0620). Significant reductions (P≦0.05) in thepercentage of piglets per gilt positive for viremia indicate reducedvertical transmission of virulent PRRSv from vaccinated gilt tooff-spring when immunized with either dose of EU PRRS 94881 MLV. Inaddition, the high titer group had a median qPCR piglet value per giltof 3.00 log₁₀ GE/ml on the DOF; while the challenge control group had amedian qPCR piglet value per gilt of 6.40 log₁₀ GE/mL in serum/bodyfluids (P=0.0030). No significant difference was detected between thelow dose group and the challenge control group for piglet viral load onDOF (P=0.0620). This data further supports the efficacy of the high doseof PRRS 94881 MLV when administered to gilts and sows.

The low titer and high titer groups exhibited means of 32.5% and 33.4%,respectively, for piglets per litter with clinical disease (a clinicalobservation score of >0) for least one day from DOF+1 to DOF+20. Theseresults were significantly lower than for the challenge control group,which exhibited a mean of 91.6% piglets per litter for the sameparameter (P≦0.0001), further supporting vaccine efficacy for both doselevels.

No significant difference was detected between groups for piglet meanbody weights on DOF 0 (P≧0.2972); while both vaccine groups hadsignificantly higher body weights on DOF+20 and ADWG from DOF 0 toDOF+20 (P≦0.0028). Once again, these results support the efficacy ofboth doses of PRRS 94881 MLV.

Necropsy results confirmed the correct categorization of almost all fetiat farrowing. Due to the very small number of feti that were listed ascrushed that were actually stillborns and stillborns that were actuallycrushed at farrowing, in comparison to the overall number of feticorrectly categorized at farrowing, no changes were made to the giltperformance data before it was analyzed. One-challenge control pigletdied subsequently to blood collection. Since this situation onlyinvolved one piglet in comparison to the large overall number of pigletsin the challenge control group, this piglet was not removed fromanalyses.

Lung samples were collected from 141, 79, 75 and 4 dead feti/pigletsfrom the challenge control, low titer, high titer, and negative controlgroups, respectively. A mean qPCR lung value of 4.68, 4.10, 3.55 and0.00 log₁₀ GE/mL was determined for the challenge control, low titer,high titer and negative control groups, respectively. No analyses wereconducted on these data since piglets alive at 20 days of age were notnecropsied, but these results highlight that gilts vaccinated with PRRS94881 MLV resulted in lower viral load in the lungs of piglets whengilts were challenged with a virulent PRRSv.

In conclusion, results from this study demonstrated significantly higherpercentages of live piglets per litter at farrowing (P≦0.0455) andhigher percentages and numbers of piglets per litter at weaning(P≦0.0203) for both vaccine groups in comparison to the challengecontrol group. Thus, the study objective was met and data from thisstudy establishes the MID of PRRS 94881 MLV in gilts as 1×10^(2.43)TCID₅₀/2 mL. These results were achieved 118 days after vaccination,which in addition establishes duration of immunity (DOI) in gilts ofapproximately 4 months.

When supportive data was examined, the high dose of PRRS 94881 MLV(1×10^(3.90) TCID₅₀/2 mL) was associated with a higher percentage andnumber of healthy piglets per gilt at farrowing (P≦0.0211), a lowerpercentage and number of weak and mummified feti (P≦0.0090), a lowerpercentage of qPCR positive gilts and lower viral load in giltspost-challenge on D125, DOF 0 and DOF+13 (P≦0.0155), a lower percentageof piglets per gilt qPCR positive and lower piglet viral load on DOF 0(P≦0.0030), a lower percentage of piglets per gilt with clinical disease(P<0.0001), and higher piglet body weights on DOF+20 and ADWG(P<0.0013).

The low dose group was associated with a higher percentage of healthypiglets per gilt at farrowing (P=0.0138), a lower percentage and numberof mummified feti (P≦0.0190), a lower percentage of qPCR positive giltsand lower viral load in gilts post-challenge on D125, D132, DOF 0 andDOF+13 (P≦0.0290), a lower percentage of piglets per gilt qPCR positiveon DOF 0 (P=0.0381), a lower percentage of piglets per gilt withclinical disease (P<0.0001), and higher piglet body weight on DOF+20 andADWG (P<0.0028).

Example 7 Evaluation of the Onset of Immunity PRRS 94881 MLV inSusceptible Piglets Following Challenge with a Heterologous EuropeanIsolate of PRRS at Two Weeks Post-Vaccination

The objective of this vaccination-challenge study was to assess theonset of immunity (OOI) two weeks after the administration of thevaccine candidate Porcine Reproductive and Respiratory Syndrome,European-derived Isolate 94881, Modified Live Virus (PRRS 94881 MLV) to14±3 days of age susceptible piglets. The primary efficacy criterion tosatisfy an OOI of 2 weeks post vaccination was if the vaccinate group(Group 1) demonstrated a significant difference (p≦0.05) for lunglesions post-challenge compared to the unvaccinated challenge controlgroup (Group 2). Secondary parameters included clinical assessmentsafter vaccination, clinical observations after challenge, rectaltemperatures, average daily weight gain, assessment of PRRS antibodiesand viremia in serum samples and quantitation of PRRS virus in lungsamples collected at necropsy.

Piglets were randomly assigned to either Group 1 (PRRS 94881 MLV-vaccinecontaining 1×10^(3.82) TCID₅₀/mL and challenged; n=20), Group 2 (placebovaccine and challenged; n=20) or Group 3 (placebo vaccine and notchallenged; n=10). Piglets were housed in plastic pens with raisedfloors (n=5/pen). Each treatment group was housed in a different room toavoid transmission of PRRSv through mechanical routes, includingaerosolization.

All animals assigned to this study completed the study. No adverseevents were reported during this study. The mean lung lesion scores onD24 were 27.4% and 54.8% for the PRRS 94881 MLV-vaccinated pigs and thechallenge controls, respectively. The mean lung lesion score for thePRRS 94881 MLV-vaccinated pigs was significantly lower than thechallenge controls (p=0.0002), and therefore the primary efficacyvariable was met and the OOI was established at 2 weeks following asingle vaccination. A significantly higher proportion of PRRS 94881MLV-vaccinated pigs had positive PRRS-antibody titers on D14, D17 andD21 compared to challenge controls (p≦0.0012). The mean AUC for viremiawas significantly lower for PRRS 94881 MLV-vaccinated pigs compared tochallenge controls for D17-D24 (50.72 and 54.61 log₁₀ GE/mL,respectively; p=0.0039) post challenge. PRRS 94881 MLV-vaccinated pigsexhibited no signs of lethargy (0%) after challenge compared with 45% ofthe challenge control pigs (p=0.0012). PRRS 94881 MLV-vaccinated pigshad higher weight gains during the post-challenge phase (SD14-SD24) ofthe study compared to challenge controls (0.3 and 0.1 kg, respectively;p=0.0003).

The significant (p≦0.05) reduction of the lung lesions, clinical signs,replication of the virus in the blood and lungs post-challenge as wellas the improvement of the growth performances in vaccinated animalsdemonstrate vaccine efficacy against virulent PRRSv when the challengeis performed 2 weeks post vaccination. It therefore supports thedemonstration of an onset of immunity of at least 2 weekspost-vaccination with PRRS 94881 MLV.

Objectives/Purpose of Study

The objective of this vaccination-challenge study was to assess theonset of immunity (OOI) two weeks after the administration of thevaccine candidate Porcine Reproductive and Respiratory Syndrome,European-derived Isolate 94881, Modified Live Virus (PRRS 94881 MLV) to14±3 days of age susceptible piglets. The primary efficacy criterion tosatisfy an OOI of 2 weeks post vaccination was if the vaccinate group(Group 1) demonstrated a significant difference (p≦0.05) for decreasedlung lesions post-challenge compared to the unvaccinated, challengecontrol group (Group 2).

The secondary efficacy parameters analyzed between the vaccine group andthe challenge control group included clinical assessmentspost-vaccination, PRRS serology, PRRS viremia post-challenge, clinicalobservations post-challenge, average daily weight gain (ADWG), rectaltemperatures and lung PRRSv quantitation.

A negative control group (Group 3), which was not vaccinated orchallenged, was included in the study to demonstrate the source herd wasfree of PRRSv infection throughout the trial period and that biosecuritywas not breached during this trial.

Schedule of Events

TABLE 7.1 Schedule of Events Study Day Dates Key Study Event −8 14 Dec.2009 Screen for negative PRRS ELISA status −1 21 Dec. 2009 Arrival atVRI; Health Exam −1 to 12 21 Dec. 2009 to Clinical Assessments 03 Jan.2010 0 22 Dec. 2009 Collect body weights Vaccinate Group 1 with IVP,Vaccinate Groups 2 & 3 with CP 7 29 Dec. 2009 Blood sample 13 to 24 04Jan. 2010 to Clinical Observations and Rectal 15 Jan. 2010 Temperatures14 05 Jan. 2010 Collect body weights and blood sample; Challenge Groups1 & 2 with heterologous European PRRS isolate 17 and 21 08 Jan. 2010 andBlood sample 12 Jan. 2010 24 15 Jan. 2010 Euthanize and necropsy pigsafter data and sample collection; Score lungs for pathology; collectlung tissues

Study Design

TABLE 7.2 Study Design Challenge on Number D14 with 1 mL/ Euthanize ofnostril and 1 mL and Piglets Treatment on D0 IM of PRRSv Necropsy Groupon D0 (14 ± 3 days of age) 205817 on D24 1 20 1.0 mL IM of Yes Yes IVP(1 × 10^(3.82) TCID₅₀/mL) 2 20 1.0 mL IM of Control Yes Yes Product (CP;Placebo matched product without PRRS 94881 MLV) 3 10 1.0 mL IM of CP NoYes

Blinding Criteria

The Study Investigator and designees were blinded to the assignedtreatment groups throughout the in-life phase of the study. To maintainthis blinding, an individual who did not participate in assessments ofthe pigs (i.e., clinical assessments, clinical observations ornecropsies) performed the randomization and administered the assignedIVP and CP treatments on D0. BIVI laboratory personnel were blinded tothe treatment each pig received while conducting their respective tasks.

Materials

Investigational Veterinary Product (IVP) and Control Product (CP)

TABLE 7.3 IVP Generic Product Name: Porcine Reproductive and RespiratorySyndrome, Modified Live Virus Strain: 94881 Production andBIVI-Production produced PRRS 94881 MLV, Lot 390-005 Formulation:(Appendix 4) in accordance with Outline of Production, Code 19S1.U_ andEU Dossier Part 2b. On D0, BIVI-Ames reconstituted/diluted PRRS 94881MLV vaccine Lot 390-005 (Appendix 4) with Phosphate buffered saline(PBS; Lot 809-003, Appendix 5) to formulate the IVP, Lot No. 257-086.Transcribed formulation records for the IVP are presented in Appendix 7(original records available upon request). Manufacturer: BoehringerIngelheim Vetmedica, Inc. 2621 North Belt Highway St. Joseph, MO 64506,USA Lot No.: N257-086 Expiry Date: An expiration date of 22 Dec. 2009was assigned to the IVP for study purposes only. Storage Conditions:Lyophilized vaccine: 2-8° C. Rehydrated/diluted IVP: 2-8° C. or on iceTesting: Batch 390-005 was tested by BIVI-QC in accordance with draftOutline of Production and EU dossier Part 2F. At the start and end ofthe vaccination procedure, BIVI-Ames personnel were contacted. BIVI-Ameslaboratory personnel tested pre- and post-vaccination aliquots for theIVP for virus titer in accordance with the PRRSv Titer Procedure(Appendix 1, Attachment 6). Test Results: Serial 390-005: Results weresatisfactory (Appendix 4). IVP Lot N257-086: Mean titer of 1 × 10^(3.82)TCID₅₀/mL (Appendix 7). IVP IVP was formulated for this study only andwas not retained. Retention:

TABLE 7.4 CP Generic Product Placebo Name: Formulation: BIVI-Productionproduced lyophilized placebo product containing inert material comprisedin the vaccine serial without PRRS 94881 MLV (Lot N240-191-062409,Appendix 6). On D0, BIVI-Ames reconstituted Lot N240-191-062409 withPhosphate buffered saline (PBS; Lot 809-003, Appendix 5) to formulatethe CP, Lot No. 257-085. Transcribed formulation records for the CP arepresented in Appendix 7 (original records available upon request).Manufacturer: Boehringer Ingelheim Vetmedica, Inc. 2621 North BeltHighway St. Joseph, MO 64506, USA Lot Number: N257-085 Expiry Date: Anexpiration date of 22 Dec. 2009 was assigned to the CP for studypurposes only. Storage Conditions: Lyophilized vaccine: 2-8° C.Rehydrated CP: 2-8° C. or on ice Testing: CP was tested by BIVI-QC forEP sterility in accordance with Special Outline No. 96 (Appendix 1,Attachment 5). Test Results: CP was determined to be sterile (Appendix7). CP Retention: CP was formulated for this study only and was notretained.

Challenge Material

TABLE 7.5 Challenge Material Name/number of PRRS isolate 205817 isolateLocation and date The European PRRS virus isolate 205817 was derivedfrom isolate of isolation incl. 190136 originally obtained from lungtissue of a newborn piglet from clinical symptoms a farm showing typicalreproductive signs of PRRS (abortions in sows and weakness in newbornpiglets) during an outbreak in Lower Saxony, Germany, in April 2004. Theattending veterinarians submitted the lung samples to bioScreen (samplearrived on 21 Apr., 2004) for diagnostic testing. Isolate #190136 wasdirectly propagated on MA 104 cells and a pure culture challenge stockwas prepared for use in future BIVI clinical trials. A pure culture ofisolate 190136 was used to inoculate pigs for evaluation of its abilityto reproduce PRRS-specific respiratory disease in a controlled,laboratory trial. Challenged animals exhibited respiratory distress andrevealed evidence of interstitial pneumonia upon histopathologicalexamination. PRRS virus was successfully re-isolated from lung lesionswas given the isolate designation 205817. Isolate 205817 was directlypropagated on MA104 cells and a pure culture challenge stock wasprepared for use in future BIVI clinical trials. Formulation: Challengevirus was thawed and diluted with MEM (Minimum Essential Medium) to atargeted titer of approximately 1 × 10⁶ TCID₅₀/3 mL on D14. An adequatevolume of challenge material was prepared. Two aliquots were removedfrom challenge material. Lot Number: N257-093 Manufacture: BoehringerIngelheim Vetmedica, Inc.-USA Storage conditions Bulk challenge materialwas stored at −70 ± 10° C. Once prepared, diluted challenge material wasmaintained on ice until it was administered. Testing: At the start andend of the challenge procedure, BIVI-Ames was contacted. BIVI-Ameslaboratory personnel tested pre- and post- challenge aliquots for virustiter in accordance with the PRRSv Titer Procedure Test Results: Thechallenge material had a mean titer of 1 × 10^(4.71) TCID₅₀/3 mL doseAdministration 1.0 mL/nostril and 1.0 mL IM in the left neck(administered to all pigs route in Groups 1 and 2 on D14). Challengematerial Challenge material was formulated for this study only and wasnot retention: retained.

Treatments

Dosing Justification

The IVP was administered as a 1.0 mL dose to assigned pigs to evaluateOOI of PRRS 94881 MLV at 2 weeks post-vaccination. The CP wasadministered as a 1.0 mL dose to Groups 2 and 3 as a placebo vaccine.

Dosing Regimen

IVP or CP was administered to an assigned pig in the right neck regionIM on D0 using a sterile 3.0 mL Luer-lock syringe and a sterile 20 g×1inch (2.54 cm) or 18 g×¾ inch (1.91 cm) needle by a person notcollecting study data. The dosing regimen is shown below in Table 7.6.

TABLE 7.6 Dosing Regimen Group Number Treatment Dose/Route Study Day 120 IVP 1.0 mL IM D0 2 20 CP 1.0 mL IM D0 3 10 CP 1.0 mL IM D0

Animal Information

Details of Study Animals

TABLE 7.7 Animal Information Source: Wilson Prairie View Farm N5627Highway DD Burlington, WI 53105 USA Number of 50 piglets: Arrival date:Pigs arrived at the Veterinary Resources, Inc. (VRI) Cambridge facilityon 21 Dec. 2009 (D-1). Arrival The 50 pigs assigned to the study wereadministered EXCEDE ® at treatment: label dose IM in the right ham afterarrival. Identification: Individually ear tagged with unique numberSpecies: Porcine Breed: Commercial crossbred Gender: Mixed (females andcastrated males) Age range: 11 to 17 days of age on D0 Weight range: 3.2to 5.5 to kg on D0 Ownership of Boehringer Ingelheim Vetmedica, Inc.test animals: Physiological On D-1, pigs selected for assignment to thestudy were observed by status: the Study Investigator and determined tobe in good health and nutritional status. Observations were recorded onthe Animal Health Examination Record form. Group-Pig Group 1 (n = 20):Group 2 (n = 20): Group 3 (n = 10): Assignments 55, 56, 60, 72, 75, 57,61, 62, 68, 78, 51, 69, 80, 85, 104, 76, 77, 83, 87, 91, 81, 86, 89, 97,110, 105, 128, 131, 133 99, 102, 116, 117, 129, 132, 135, 150, and 155124, 141, 142, 144, 152, 154, 160, 165, 156 and 162 167 and 168

Inclusion/Exclusion Criteria

All piglets enrolled in this study were PRRS ELISA negative and werehealthy at the time of vaccination as determined by observation.

Post-Inclusion Removal Criteria

No pigs were removed from the study.

Animal Management and Housing

Animal Housing

Piglets were housed at Veterinary Resources, Inc. (VRI) in Cambridge,Iowa for the duration of the study. Groups 1, 2 and 3 were housed inuniform but separate rooms to ensure biosecurity. Piglets were housed inmultiple pens (5 piglets/pen) within each room. Group 1 was housed in 4pens in Room 5, Group 2 was housed in 4 pens in Room 6 and Group 3 washoused in 2 pens in Room 4. Pens consisted of plastic tubs on raisedstands with plastic slatted flooring. Each pen contained a plastic6-hole feeder and a nipple waterer. Each isolation room was constructedidentical to the others and all are biohazard level 2 (BL2) compliant,hepafiltered, mechanically ventilated with thermostat regulatedtemperature control.

Treatment group isolation was necessary in this study as it is wellknown within the scientific community that PRRSv readily spreads frompig to pig via various mechanisms including aerosolization. Thisincludes avirulent live PRRS vaccines as these biological productsinclude attenuated virus particles that mimic the characteristics ofvirulent wild-type PRRS without the capability to cause disease. Propermethods were in place to ensure that biosecurity was maintained and thatvaccinated animals did not accidentally cross-contaminatenon-vaccinated, PRRSv naïve negative control animals. Appropriatemeasures were taken by test facility staff to adequately clean anddisinfect each room prior to its usage for this study.

Each room in the facility has fans and heaters to aid in sufficient aircirculation and heating. The ventilation system is separate yetidentical for each room, so air is not shared between rooms.

Solid feed was stored in bags, free from vermin. Water was ad libitum.Piglets were fed a commercial ration (Lean Metrics Infant, Purina Mills,St. Louis, Mo.) medicated with tiamulin (35 gm/ton) andchlortetracycline (400 gm/ton) ad libitum appropriate for their size,age, and condition; according to acceptable animal husbandry practicesfor the region.

The pigs were in good health and nutritional status before initiation ofthe study as determined by the Study Investigator.

During the study, select animals were observed with mild loss of bodycondition, rough haired appearance, swollen joints and varying degreesof lameness. The Study Investigator considered all of these to benon-specific conditions that commonly occur in groups of pigs housed inconfinement. Coughing, sneezing, rapid respiration, dyspnea and mild tomoderate lethargy were also noted in select pigs after challenge andwere considered typical clinical signs associated with pneumonia,although non-specific for etiology. The Study Investigator determinedthat concomitant treatments were not required for any animals duringthis study.

All pigs assigned to this study were disposed of by commercialincineration after euthanasia and necropsy on D24. No food products fromanimals enrolled in this study entered the human food chain.

Assessment of Efficacy

To assess the OOI of PRRS 94881 MLV at 2 weeks post-vaccination, Groups1 & 2 were challenged on D14 and lung lesions post-challenge wereevaluated. An OOI of 2 weeks post-vaccination was achieved if Group 1(minimum immunizing dose of PRRS 94881 MLV) demonstrated significantlydecreased (p≦0.05) lung pathology post-challenge compared with thechallenge control group (Group 2).

The secondary efficacy parameters analyzed between the vaccine group andthe challenge control group included clinical assessments aftervaccination, clinical observations after challenge, rectal temperatures,body weight and average daily weight gain (ADWG), assessment of PRRSantibodies and viremia in serum samples and quantitation of PRRS virusin lung samples collected at necropsy.

A negative control group (Group 3), which was not challenged, wasincluded in the study to demonstrate the source herd was free of PRRSinfection and that biosecurity was maintained throughout the study.

Criteria for a Valid Test

Pre-purchase and D0 serum samples were all required to be negative forPRRS antibodies.

Serum samples collected from Groups 2 and 3 up to the day of challengeand from Group 3 until study completion had to be free of PRRSantibodies for the study to be valid.

Primary Outcome Parameter

The primary efficacy variable for statistical evaluation was total lunglesion scores at D24 of the study.

Total Lung Lesion Scores

On Day 24 after data and samples were collected and recorded, all studypigs were euthanized following VRI SOP PRC1027 (Appendix 1, Attachment8). Each pig was necropsied in accordance with VRI SOP PRC 1028 Thethoracic cavity was exposed by a designee and the heart and lungs wereremoved. The Study Investigator examined each set of lungs, describedany gross pathology noted and determined the % pathology for each lunglobe. Observations and data were recorded on the Necropsy Report Recordform. A total lung lesion score was determined for each pig by using theEP formula.

Supportive Parameters

Other parameters to be analyzed between Group 1 and Group 2 includedclinical assessments post-vaccination, PRRS serology, viremiapost-vaccination, clinical observations post-challenge, ADWG, rectaltemperatures and lung virus quantitation post challenge. Theseparameters were analyzed as supportive parameters and did not serve asprimary parameters to satisfy the study objective.

Clinical Assessment

All pigs were observed on the days outlined in Table 7.1 for clinicalassessments post-vaccination by the Study Investigator or designees.Observations were recorded on the Clinical Assessment Record form.

PRRS Serology

Venous whole blood was collected on the days outlined in Table 3.Briefly, approximately 2-5 mL of blood was collected from each pigletinto an appropriate sized serum separator tube (SST). Sample collectionswere recorded on the Sample Collection Record form. Blood in SSTs wasallowed to clot at room temperature. Blood samples were delivered toBIVI-Ames on the day of collection and Specimen Delivery Record form wascompleted. Blood samples were spun down by BIVI-Ames and serum washarvested, split and transferred to appropriate tubes. Each tube waslabeled with the piglet's ID number, the study number, the date ofcollection, the study day and the sample type. At BIVI-Ames, one set ofserum samples was held at 2-8° C. and the other set of serum samples washeld at −70±10° C.

The serum samples collected days 0, 7, 14, 17, 21 and 24 and held at2-8° C. were tested by BIVI-Ames for PRRS antibodies. Results werereported as negative (ELISA S/P ratio of <0.4) or positive (ELISA S/Pratio of ≧0.4).

PRRS Viremia

The other set of serum samples collected on days 0, 7, 14, 17, 21 and 24and held at −70±10° C. at BIVI-Ames until the in-life phase of the studywas completed.

A completed Specimen Delivery Record form was included with theshipment. bioScreen tested serum samples for PRRSv RNA by qPCR. Resultswere reported as genome equivalent/mL (log GE/mL).

Clinical Observations Post-Challenge

Piglets were observed for clinical signs of disease on the days outlinedin Table 7.1. Observations were conducted by the Study Investigator ordesignees and were recorded on the Clinical Observation Record form.Piglets were observed each day for respiration, behavior and cough basedon the clinical observation scoring system outlined below in Table 7.8.

TABLE 7.8 Clinical Observation Scoring System Respiration Behavior Cough0 = normal respiration 0 = normal 0 = none 1 = panting/rapid 1 = mild tomoderate 1 = soft or intermittent respiration lethargy cough 2 = dyspnea2 = severely lethargic 2 = harsh or severe, 3 = dead or recumbentrepetitive cough 3 = dead 3 = dead

Average Daily Weight Gain (ADWG)

Individual body weights were collected on the days outlined in Table 3.Each pig was weighed on a calibrated scale by the Study Investigator ordesignees. Results were reported in kg on the Body Weight Record form.Average daily weight gain was determined from the D0 to D14 and from D14to D24.

Rectal Temperatures

Rectal temperatures were collected by the Study Investigator ordesignees on the days outlined in Table 6.1. Rectal temperatures wererecorded in ° C. on the Clinical Observation Record form.

PRRS Virus Quantitation in Lung Tissue

For each set of lungs, two samples from the Left and Right Apical lobes,the Left and Right Cardiac lobes, the Left and Right Diaphragmatic lobesand the Intermediate lobe, were retained. Each lung sample wasapproximately 1 inch (2.54 cm)×1 inch (2.54 cm). For one set of lungsamples, all three samples from the left side were combined into onecontainer; while all three samples from the right side and theIntermediate lung lobe sample were combined into another container. Eachcontainer was filled with a sufficient amount of 10% formalin solution.For the other set of lung samples, all three lung samples from the leftside were combined into one WHIRLPAK®; while all three samples from theright side and the Intermediate lung lobe sample were combined intoanother WHIRLPAK®. All containers and WHIRLPAKS® were appropriatelylabeled with animal number, study number, date of collection, study day,sample type and whether the samples are from the left or right side.Lung samples in WHIRLPAKS® were stored on dry ice until transported toBIVI-Ames while samples in formalin were stored at room temperature.Sample collections were recorded on the Necropsy Report Record form.Formalin fixed lung tissue samples and WHIRLPAK® lung samples weretransferred to BIVI-Ames. A completed Specimen Delivery Record form wasincluded with each shipment.

A completed Specimen Delivery Record form was included with theshipment. bioScreen tested lung samples for PRRSv RNA by qPCR (Appendix1, Attachment 7). Left lung tissues were homogenized and tested. Rightlung tissues and intermediate lung lobe samples were homogenized andtested. Results were reported as genome equivalent (log GE/mL) for leftand right lung samples.

Adverse Events

No adverse events were reported during this study.

Statistical Methods

Experimental Unit

Treatment groups had to be housed in separate rooms in this study toavoid transmission of PRRSv to non-vaccinated groups. Therefore, roomwas the experimental unit. However, for the purposes of this analysis,possible bias due to confounding “room” and “treatment” effects wereignored, and piglet was used as the experimental unit.

Randomization

Fifty (50) piglets were blocked by weight (n=5 piglets/block). Each pigwas assigned a random number using the random number function in Excel.Within each weight block, pigs were ranked in ascending numerical orderof the assigned random number. The treatment groups were then assignedto pigs in this numerical order: the 2 lowest random numbers wereassigned to Group 1, the next 2 numbers were assigned to Group 2 and thehighest number was assigned to Group 3. Groups 1 & 2 each contained 20pigs and Group 3 contained 10 pigs.

Analysis

The statistical analyses and data summaries were conducted by Dr. rer.hort. Martin Vanselow, Biometrie & Statistik, Zum Siemenshop 21, 30539Hannover, Germany, +49(0) 511 606 777 650, m.vanselow@t-online.de.

Data were analyzed assuming a completely random design structure. Thestatistical analyses were performed using SAS software release 8.2 (SAS,Cary, USA/North Carolina, SAS Institute Inc. All tests on differenceswere designed as two-sided tests at α=5%.

Total Lung Lesion Scores

The total lung lesion score on the day of necropsy (D24) was measured asthe percentage of lung involvement calculated according to the weightingformula recommended in the draft monograph Porcine Enzootic PneumoniaVaccine (inactivated). This formula takes into account the relativeweight of each of the seven lung lobes. The assessed percentage of lunglobe area with typical lesions was multiplied by the respective factorper lung lobe giving the total weighted lung lesions score. The factorsfor the respective lung lobes are presented in Table 7.9.

TABLE 7.9 Factors for Calculating Lung Lesion Scores Lung lobe FactorLeft apical 0.05 Left cardiac 0.06 Left diaphragmatic 0.29 Right apical0.11 Cardiac 0.10 Right diaphragmatic 0.34 Right accessory/intermediate0.05

The treatment groups were compared on differences using the WilcoxonMann-Whitney test.

Clinical Assessment Post-Vaccination

Frequency tables of animals with at least one positive finding betweenD1 and D12 were generated. Differences between treatment groups weretested by Fisher's exact test.

PRRS Serology

Frequency tables of positive ELISA results were generated. Differencesbetween treatment groups were tested by Fisher's exact test.

PRRS Viremia

The viremia data were evaluated separately for each day ofinvestigation. Additionally, for viral load the areas under theindividual response curves between D14 and D24 (AUC D14-D24) and betweenD17 and D24 (AUC D17-D24) were analyzed.

The quantitative PCR data (PRRS viral load [log₁₀ GE/mL]) were used forcomparisons between the treatment groups by the Wilcoxon Mann-Whitneytest. Prior to the calculations the analytical result ‘not detected’ wasreplaced by a log₁₀ GE/mL value of 0.0 and ‘positive’ was replaced by3.0. The treatment groups were tested on differences using the WilcoxonMann-Whitney test.

Clinical Observations Post-challenge

Frequency tables of animals with at least one positive finding betweenD15 and D24 were generated. Differences between treatment groups weretested by Fisher's exact test.

The maximum scores and the mean scores per animal from D15 to D24 forrespiration, behavior, coughing and for all three added together (total)were used for the statistical evaluation. Differences between treatmentgroups were tested by the Wilcoxon Mann-Whitney test.

Body Weight and Average Daily Weight Gain

Individual daily weight gains were calculated for the time periodsbetween D0 and D14 and between D14 and D24. For each day ofinvestigation and for each time period descriptive statistics werecalculated. Differences between treatment groups were tested usinganalysis of variance and subsequent t-tests. Least squares means of thegroups and differences between least squares means with 95% confidenceintervals were calculated from the analysis of variance.

Rectal Temperatures

Differences between treatment groups with respect to the originaltemperature data were tested using analysis of variance and subsequentt-tests. Least squares means of the groups and differences between leastsquares means with 95% confidence intervals were calculated from theanalysis of variance.

PRRS Virus Quantitation in Lung Tissues

The quantitative PCR data (PRRS viral load [log₁₀ GE/mL]) from lungscollected on D24 were used for comparisons between the treatment groupsby the Wilcoxon Mann-Whitney test. The average (log₁₀ GE/mL) of the leftand right lung qPCR results were used for the evaluation. Prior to thecalculations the analytical result ‘not detected’ was replaced by log₁₀GE/mL of 0.0 and ‘positive’ was replaced by 3.0.

Frequency tables of positive qPCR results were generated. Differencesbetween treatment groups were tested by Fisher's exact test.

Results

Total Lung Lesion Scores

A summary of the group total lung lesion scores and the associatedp-value is shown below in Table 7.10.

TABLE 7.10 Total Lung Lesion Scores (%) Group¹ N Min. Max. Median 95% CIQRange Mean p value 1 20 0.06 59.30 27.550 12.270 40.600 29.515 27.3680.0002 2 20 13.86 91.60 55.200 47.300 66.500 21.850 54.841 3 10 0.000.06 0.000 0.000 0.000 0.000 0.006 NI ¹Group 1 = PRRS 94881 MLV vaccineat MID, challenged; Group 2 = placebo-treated, challenged; Group 3 =placebo-treated, not challenged. NI = Not included in statisticalanalysis.

Mean piglet D24 total lung lesion scores were 27.368% and 54.841% forthe PRRS 94881 MLV-vaccinated group and challenge controls,respectively. The lesion score for the PRRS-vaccinated pigs wassignificantly lower than the mean lesion score for the challengecontrols (p=0.0002).

PRRS Viremia

A summary of the PRRSv RNA detected in serum by qPCR data is shown belowin Table 7.11.

TABLE 7.11 PRRSv RNA Detected by qPCR in Serum (log₁₀ GE/mL) by Day DayGroup¹ N Min. Max. Median 95% CI QRange Mean p value  7 1 20 0.00 5.343.00 3.00 3.79 0.82 3.17 <0.0001   2 20 0.00 0.00 0.00 0.00 0.00 0.000.00 3 10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI 14 1 20 0.00 4.29 3.323.00 3.77 0.84 3.30 <0.0001   2 20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 310 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI 17 1 20 5.54 8.07 6.72 6.477.08 0.80 6.78 <0.0001   2 20 6.44 9.02 8.18 7.47 8.47 1.09 8.00 3 100.00 0.00 0.00 0.00 0.00 0.00 0.00 NI 21 1 20 6.18 8.73 7.38 7.13 8.080.98 7.51 0.0565 2 20 7.22 8.86 7.87 7.62 8.11 0.57 7.88 3 10 0.00 0.000.00 0.00 0.00 0.00 0.00 NI 24 1 20 5.82 8.54 7.15 6.73 7.84 1.16 7.260.6251 2 20 6.53 8.29 7.27 6.97 7.60 0.67 7.34 3 10 0.00 0.00 0.00 0.000.00 0.00 0.00 NI AUC 1 20 56.95 78.02 65.10 60.39 70.05 9.76 65.840.4945 14-24 2 20 58.74 74.30 67.02 64.38 68.24 4.83 66.61 3 10 0.000.00 0.00 0.00 0.00 0.00 0.00 NI AUC 1 20 42.98 59.51 49.52 47.46 54.307.14 50.72 0.0039 17-24 2 20 49.08 60.99 54.35 52.93 55.38 3.63 54.61 310 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI ¹Group 1 = PRRS 94881 MLVvaccine at MID, challenged; Group 2 = placebo-treated, challenged; Group3 = placebo-treated, not challenged. NI = Not included in statisticalanalysis. AUC = Area under the curve; GE/ml per day

PRRSv RNA was not detected in the serum of any piglets on D0. PRRS 94881MLV-vaccinated pigs had mean values of 3.17 and 3.30 log₁₀ GE/mL on D7and D14, respectively. The values were significantly higher thanchallenge controls on both of these days (p<0.0001), as challengecontrols did not have any PRRSv RNA detected until D17. On that day,mean values were 6.78 and 8.00 log₁₀ GE/mL for PRRS 94881 MLV-vaccinatedpiglets and challenge controls, respectively. The D17 value forchallenge controls was significantly higher than the PRRS 94881MLV-vaccinated piglets (p<0.0001). Mean values for PRRS 94881MLV-vaccinated pigs on D21 and D24 were 7.51 and 7.26 log₁₀ GE/mL on D21and D24 respectively, compared to 7.88 and 7.34 log₁₀ GE/mL forchallenge controls on the same days. There were no significantdifferences between PRRS 94881 MLV-vaccinated pigs on D21 or 24(p≧0.0565). No PRRSv RNA was detected in serum from any negative controlpig during this study.

There were no differences between the AUC 14-24 for PRRS 94881MLV-vaccinated pigs and challenge controls pigs (65.84 and 66.61,respectively; p=0.4945). PRRS 94881 MLV-vaccinated pigs had asignificantly lower AUC for D17-D24 compared to challenge controls(50.72 and 54.61, respectively; p=0.0039).

PRRS Virus Quantitation in Lung Tissues

Individual PRRSv qPCR results from lung tissues collected at necropsy onD24 are presented in Addendum 1, Table 30. A summary of the PRRSv RNAdetected in lung tissues by qPCR data is shown below presented in Table7.12 and a summary of the frequency of animals with positive qPCR atnecropsy is shown below in Table 7.13.

TABLE 7.12 Lung Viruse Isolation, qPCR (mean log10 GE/mL) at Necropsy(D24) Group¹ N Min. Max. Median 95% CI QRange Mean p value 1 20 6.638.26 7.46 7.07 7.86 0.84 7.47 0.0101 2 20 6.55 8.67 7.99 7.69 8.14 0.547.88 3 10 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI ¹Group 1 = PRRS 94881MLV vaccine at MID, challenged; Group 2 = placebo-treated, challenged;Group 3 = placebo-treated, not challenged. NA = Not applicable becauseof lack of variability. NI = Not included in statistical analysis.

TABLE 7.13 Frequency of Animals with Possible PRRSv RNA aPCR from LungTissues Collected at Necropsy (D24) Day Group N % 95% CI Total P 24 1 20100 83.2 100.0 20 NA 2 20 100 83.2 100.0 20 3 0 0 0.0 30.8 10 NI ¹Group1 = PRRS 94881 MLV vaccine at MID, challenged; Group 2 =placebo-treated, challenged; Group 3 = placebo-treated, not challenged.NA = Not applicable because of lack of variability. NI = Not included instatistical analysis.

PRRSv RNA was detected in the lung tissues of all piglets in both thePRRS 94881 MLV-vaccinated group and all piglets in the challenge controlgroup. There was no difference between these groups. PRRSv RNA was notdetected in the lung samples of any negative control piglets.

Clinical Observations Post-challenge

The frequency of piglets with at least one positive clinical assessmentscore in the post-challenge period (D15-D24) is shown below in Table7.14.

TABLE 7.14 Frequency of Piglets with a Positive Clinical ObservationPost Challenge (D15-D24) N % Parameter Group¹ positive positive 95% CITotal p value Respiration 1 2 10 1.2 31.7 20 0.2351 2 6 30 11.9 54.3 203 0 0 0.0 30.8 10 NI Behavior 1 0 0 0.0 16.8 20 0.0012 2 9 45 23.1 68.520 3 0 0 0.0 30.8 10 NI Coughing 1 6 30 11.9 54.3 20 0.2003 2 11 55 31.576.9 20 3 0 0 0.0 30.8 10 NI Total 1 6 30 11.9 54.3 20 0.0562 2 13 6540.8 84.6 20 3 0 0 0.0 30.8 10 NI ¹Group 1 = PRRS 94881 MLV vaccine atMID, challenged; Group 2 = placebo-treated, challenged; Group 3 =placebo-treated, not challenged. NI = Not included in statisticalanalysis.

Abnormal respiration was observed in both the PRRS 94881 MLV-vaccinatedgroup (10%) and in the challenge control group (30%), however, thesevalues were not significantly different (p=0.2351).

Abnormal behavior was only observed in the challenge control group(45%), and not in the PRRS 94881 MLV-vaccinated group (0%). The PRRS94881 MLV-vaccinated group had a significantly lower incidence ofabnormal behavior than the challenge controls (p=0.0012).

Coughing was observed in both the PRRS 94881 MLV-vaccinated group (30%)and in the challenge control group (55%). These values were notsignificantly different (p=0.2003).

The percentages of piglets with total clinical scores >0 were 30% and65% for the PRRS 94881 MLV-vaccinated group and the challenge controlgroup, respectively. These values were not significantly different(p=0.0562).

No clinical signs were observed in the negative control group at anytime after challenge.

A summary of the group maximum clinical observation scores for thepost-challenge period (D15 through D24) is shown below in Table 7.15.

TABLE 7.15 Post-Challenge Maximum Clinical Scores, D15 through D24Parameter Group¹ N Min. Max. Median 95% CI QRange Mean p valueRespiration 1 20 0 1 0 0 0 0 0.1 0.1872 2 20 0 2 0 0 1 1 0.4 3 10 0 0 00 0 0 0.0 NI Behavior 1 20 0 0 0 0 0 0 0.0 0.0012 2 20 0 1 0 0 1 1 0.5 310 0 0 0 0 0 0 0.0 NI Coughing 1 20 0 1 0 0 1 1 0.3 0.1129 2 20 0 2 1 01 1 0.7 3 10 0 0 0 0 0 0 0.0 NI Total 1 20 0 1 0 0 1 1 0.3 0.0072 2 20 04 1 0 2 2 1.2 3 10 0 0 0 0 0 0 0.0 NI ¹Group 1 = PRRS 94881 MLV vaccineat MID, challenged; Group 2 = placebo-treated, challenged; Group 3 =placebo-treated, not challenged. NI = Not included in statisticalanalysis.

Abnormal respiration was observed in both the PRRS 94881 MLV-vaccinatedgroup and the challenge controls after challenge administration, withmaximum scores of 1 (panting/rapid respiration) and 2 (dyspnea),respectively. There was no significant difference between theserespiration scores (p=0.1872). The median maximum respiration score was0 for both groups.

No abnormal behavior was observed in the PRRS 94881 MLV-vaccinated groupin the post-challenge period (maximum score=0). In contrast, thechallenge control group had a maximum behavior score of 1 (mild tomoderate lethargy; p=0.0012) although the median score for this groupwas 0. The maximum score for the PRRS 94881 MLV-vaccinated group wassignificantly lower than the score for the challenge control group(p=0.0012). Median maximum behavior scores were 0 for both groups.

Coughing was observed in both the PRRS 94881 MLV-vaccinated group and inthe challenge control group after challenge. Maximum scores were 1 (softor intermittent cough) and 2 (harsh or severe, repetitive cough), andmedian scores were 0 and 1, for PRRS 94881 MLV-vaccinated and challengecontrols, respectively. There were no significant differences betweenthese groups (p=0.1129). Median maximum coughing scores were 0 and 1 forthe PRRS 94881 MLV-vaccinated group and challenge control group,respectively.

Maximum total scores were 1 and 4 and median total scores were 0 and 1for the PRRS 94881 MLV-vaccinated group and the challenge control group,respectively. The maximum score for the PRRS 94881 MLV-vaccinated groupwas significantly lower than the score for the challenge control group(p=0.0072). Median total scores were 0 and 1 for the PRRS 94881MLV-vaccinated group and challenge control group, respectively.

No clinical signs were observed from D15 through D24 in thenon-challenged negative control group during this study. This group hada maximum score of 0 for each parameter.

A summary of the group mean clinical observation scores for thepost-challenge period (D15 through D24) is shown below in Table 7.16.

TABLE 7.16 Post-Challenge Mean Clinical Scores, D15 through D24Parameter Group¹ N Min. Max. Median 95% CI QRange Mean p valueRespiration 1 20 0.0 0.2 0.00 0.00 0.00 0.00 0.02 0.1394 2 20 0.0 0.60.00 0.00 0.10 0.10 0.07 3 10 0.0 0.0 0.00 0.00 0.00 0.00 0.00 NIBehavior 1 20 0.0 0.0 0.00 0.00 0.00 0.00 0.00 0.0012 2 20 0.0 0.8 0.000.00 0.10 0.10 0.12 3 10 0.0 0.0 0.00 0.00 0.00 0.00 0.00 NI Coughing 120 0.0 0.4 0.00 0.00 0.10 0.10 0.07 0.0835 2 20 0.0 0.7 0.10 0.00 0.300.35 0.17 3 10 0.0 0.0 0.00 0.00 0.00 0.00 0.00 NI Total 1 20 0.0 0.40.00 0.00 0.10 0.15 0.08 0.0103 2 20 0.0 1.4 0.25 0.00 0.40 0.50 0.35 310 0.0 0.0 0.00 0.00 0.00 0.00 0.00 NI ¹Group 1 = PRRS 94881 MLV vaccineat MID, challenged; Group 2 = placebo-treated, challenged; Group 3 =placebo-treated, not challenged. NI = Not included in statisticalanalysis

Mean clinical observation scores followed a pattern similar to maximumclinical scores with significant differences only observed between thePRRS 94881 MLV-vaccinated group and the challenge control group for meanbehavior score (p=0.0012) and mean total score (p=0.0103).

Mean respiration scores were 0.02 and 0.07 for the PRRS 94881MLV-vaccinated group and the challenge control group, respectively. Meanbehavior scores were 0.00 and 0.12 for the PRRS 94881 MLV-vaccinatedgroup and the challenge control group, respectively. Mean coughingscores were 0.07 and 0.17 for the PRRS 94881 MLV-vaccinated group andthe challenge control group, respectively. Mean total scores were 0.08and 0.35 for the PRRS 94881 MLV-vaccinated group and challenge controlgroup, respectively.

No clinical signs were observed from D15 through D24 in thenon-challenged negative controls during this study. This group had amean score of 0 for each parameter.

Body Weight and Average Daily Weight Gain

A summary of the body weights on D0, D14 and D24 and ADWG for D0 to D14and D14 to D24 are shown below in Table 7.17.

TABLE 7.17 Body Weight and Average Daily Weight Gain (kg and kg/d)Day(s) Group¹ N Min. Max. Median Mean SD  0 1 20 3.3 5.5 3.95 4.14 0.5892 20 3.2 5.2 4.05 4.17 0.603 3 10 3.4 5.1 4.00 4.07 0.556 14 1 20 5.69.4 7.60 7.64 1.029 2 20 6.0 8.9 7.30 7.39 0.909 3 10 5.5 9.3 6.95 7.221.187 24 1 20 7.0 13.9 10.40 10.26 1.693 2 20 6.4 10.9 8.80 8.87 1.328 310 6.8 12.9 10.90 10.64 1.807 ADWG 1 20 0.164 0.343 0.2571 0.25000.05254 0-14 2 20 0.179 0.307 0.2357 0.2304 0.03939 3 10 0.150 0.3070.2071 0.2250 0.04906 ADWG 1 20 0.090 0.460 0.2600 0.2620 0.08907 14-242 20 −0.060 0.290 0.1600 0.1475 0.09060 3 10 0.130 0.440 0.3700 0.34200.10130 ¹Group 1 = PRRS 94881 MLV vaccine at MID, challenged; Group 2 =placebo-treated, challenged; Group 3 = placebo-treated, not challenged.

Mean body weights on D0 were 4.1 and 4.2 kg for the PRRS 94881MLV-vaccinated group and the challenge control group, respectively. ByD14, mean body weights were 7.6 and 7.4 kg for the PRRS 94881MLV-vaccinated group and the challenge control group, respectively. OnD24, mean body weights were 10.3 and 8.9 kg for the PRRS 94881MLV-vaccinated group and the challenge control group, respectively.Average daily weight gains (ADWG) for the vaccination period (D0 to D14)were 0.25 and 0.23 kg/d for the PRRS 94881 MLV-vaccinated group and thechallenge control group, respectively. ADWGs for the challenge period(D14 to D24) were 0.26 and 0.15 kg/d for the PRRS 94881 MLV-vaccinatedgroup and challenge control group, respectively. ADWGs for the negativecontrols were 0.23 and 0.34 kg/d for D0-D14 and D14-D24, respectively.

Negative control piglets had mean body weights of 4.1, 7.2 and 10.6 kgon D0, D14 and D28, respectively.

A summary of the LS Mean and statistical analysis of body weights andADWG for the PRRS 94881 MLV-vaccinated group and the challenge controlgroup is shown below in Table 7.18.

TABLE 7.18 LS Mean Body Weight and Daily Gain (kg) Day(s) Group¹ LS Mean95% confidence interval p value 0 1 4.14 3.865 4.405 0.8743 2 4.17 3.8954.435 Diff. 1 − 2 −0.03 −0.411 0.351 14 1 7.64 7.196 8.074 0.4297 2 7.396.951 7.829 Diff. 1 − 2 0.25 −0.376 0.866 24 1 10.26 9.566 10.944 0.00632 8.87 8.176 9.554 Diff. 1 − 2 1.39 0.416 2.364 ADWG 1 0.2500 0.228980.27102 0.1889  0-14 2 0.2304 0.20934 0.25138 Diff. 1 − 2 0.0196−0.01008 0.04937 ADWG 1 0.2620 0.22133 0.30267 0.0003 14-24 2 0.14750.10683 0.18817 Diff. 1 − 2 0.1145 0.05699 0.17201 ¹Group 1 = PRRS 94881MLV vaccine at MID, challenged; Group 2 = placebo-treated, challenged.

Day 0 LS Mean body weights were 4.14 and 4.17 kg for the PRRS 94881MLV-vaccinated piglets and the challenge control group, respectively.The difference was −0.03 kg, which was not significantly different(p=0.8743). On D14, LS Mean body weights were 7.64 and 7.39 kg for thePRRS 94881 MLV-vaccinated group and the challenge control group,respectively. The difference was 0.25 kg, which was also notsignificantly different (p=0.4297). On D24, the LS Mean body weightswere 10.26 and 8.87 for the PRRS 94881 MLV-vaccinated group and thechallenge control group, respectively. The difference on this day was1.39 kg, and the vaccinated group weight was significantly higher thanthe challenge control group (p=0.0063).

LS Mean ADWGs for the vaccination period (D0-D14) were 0.25 and 0.23kg/d for the PRRS 94881 MLV-vaccinated group and the challenge controlgroup, respectively. These values were not significantly different(p=0.1889). LS Mean ADWGs during the post-challenge period (D14-D24)were 0.26 and 0.15 for the PRRS 94881 MLV-vaccinated group and thechallenge control group, respectively. The ADWG for the PRRS 94881MLV-vaccinated group was significantly higher than the ADWG for thechallenge control group (p=0.0003).

Rectal Temperatures

A summary of rectal temperatures is shown below in Tables 7.19 and 7.20.A summary of the LS Mean and statistical analysis of rectal temperaturefor the PRRS 94881 MLV-vaccinated group and the challenge control groupis shown below in Tables 7.21 and 7.22.

TABLE 7.19 Rectal Temperature (° C.) Day 13-22 Day Group¹ N Min. Max.Median Mean SD 13 1 20 39.3 40.3 39.80 39.77 0.247 2 20 38.9 40.0 39.3539.39 0.292 3 10 39.0 39.7 39.15 39.26 0.267 14 1 20 39.4 40.2 39.7539.76 0.226 2 20 39.0 39.8 39.40 39.37 0.220 3 10 39.1 40.3 39.40 39.510.375 15 1 20 39.3 40.4 39.65 39.69 0.258 2 20 39.4 41.1 39.70 39.900.538 3 10 39.1 40.3 39.40 39.52 0.371 16 1 20 39.9 41.3 40.80 40.680.417 2 20 39.3 40.3 39.75 39.77 0.279 3 10 39.1 39.9 39.45 39.46 0.26317 1 20 39.2 40.6 39.80 39.89 0.363 2 20 39.4 40.6 39.85 39.90 0.285 310 39.2 40.0 39.50 39.53 0.226 18 1 20 39.3 41.0 39.95 39.99 0.492 2 2039.5 41.2 40.20 40.29 0.472 3 10 38.9 39.7 39.30 39.30 0.211 19 1 2039.7 41.6 40.35 40.40 0.464 2 20 39.5 41.1 40.65 40.55 0.451 3 10 39.039.6 39.20 39.22 0.199 20 1 20 39.7 41.5 40.50 40.52 0.449 2 20 39.541.5 40.65 40.61 0.531 3 10 39.1 40.1 39.40 39.49 0.281 21 1 20 39.641.1 40.30 40.22 0.413 2 20 39.4 41.0 40.10 40.12 0.371 3 10 39.2 40.239.45 39.59 0.351 22 1 20 39.8 41.0 40.20 40.34 0.391 2 20 39.6 41.240.30 40.41 0.437 3 10 39.0 40.0 39.40 39.45 0.276 ¹Group 1 = PRRS 94881MLV vaccine at MID, challenged; Group 2 = placebo-treated, challenged;Group 3 = placebo-treated, not challenged.

TABLE 7.20 Rectal Temperature (° C.) Day 23-24 Day Group¹ N Min. Max.Median Mean SD 23 1 20 39.6 41.2 40.25 40.36 0.454 2 20 39.5 41.6 40.6040.60 0.482 3 10 39.3 40.1 39.70 39.68 0.290 24 1 20 39.8 41.3 40.3040.39 0.421 2 20 39.7 41.6 40.30 40.50 0.531 3 10 39.1 40.2 39.60 39.660.389 ¹Group 1 = PRRS 94881 MLV vaccine at MID, challenged; Group 2 =placebo-treated, challenged; Group 3 = placebo-treated, not challenged

TABLE 7.21 LS Mean Rectal Temperature (° C.) Day 13-20 Day Group¹ LSMean95 % confidence interval p value 13 1 39.77 39.648 39.892 <0.0001 239.39 39.268 39.512 Diff. 1 − 2 0.38 0.207 0.553 14 1 39.76 39.65439.856 <0.0001 2 39.37 39.269 39.471 Diff. 1 − 2 0.39 0.242 0.528 15 139.69 39.494 39.876 0.1241 2 39.90 39.704 40.086 Diff. 1 − 2 −0.21−0.480 0.060 16 1 40.68 40.514 40.836 <0.0001 2 39.77 39.609 39.931Diff. 1 − 2 0.91 0.678 1.132 17 1 39.89 39.737 40.033 0.8852 2 39.9039.752 40.048 Diff. 1 − 2 −0.02 −0.224 0.194 18 1 39.99 39.767 40.2030.0528 2 40.29 40.072 40.508 Diff. 1 − 2 −0.31 −0.614 0.004 19 1 40.4040.188 40.602 0.3065 2 40.55 40.338 40.752 Diff. 1 − 2 −0.15 −0.4430.143 20 1 40.52 40.293 40.737 0.5659 2 40.61 40.383 40.827 Diff. 1 − 2−0.09 −0.405 0.225 ¹Group 1 = PRRS 94881 MLV vaccine at MID, challenged;Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, notchallenged

TABLE 7.22 LS Mean Rectal Temperature (° C.) Day 21-24 Day Group¹ LSMean95% confidence interval p value 21 1 40.22 40.037 40.393 0.4489 2 40.1239.942 40.298 Diff. 1 − 2 0.10 −0.156 0.346 22 1 40.34 40.152 40.5280.6231 2 40.41 40.217 40.593 Diff. 1 − 2 −0.07 −0.331 0.201 23 1 40.3640.143 40.567 0.1062 2 40.60 40.388 40.812 Diff. 1 − 2 −0.25 −0.5450.055 24 1 40.39 40.168 40.602 0.4526 2 40.50 40.283 40.717 Diff. 1 − 2−0.12 −0.422 0.192 ¹Group 1 = PRRS 94881 MLV vaccine at MID, challenged;Group 2 = placebo-treated, challenged; Group 3 = placebo-treated, notchallenged

Mean and LS Mean rectal temperature for the PRRS 94881 MLV-vaccinatedpiglets were 39.77° C. on the day before challenge, and ranged from39.69° C. (D15) to 40.68° C. (D16) after challenge. Mean and LS Meanrectal temperature for the challenge controls were 39.39° C. on the daybefore challenge and ranged from 39.77° C. (D16) to 40.61° C. (D20)after challenge. Least square Means rectal temperatures weresignificantly lower for challenge controls compared to PPRS 94881MLV-vaccinated piglets before challenge administration (D13 and D14) andon D16 after challenge (p<0.0001). There were no other significantdifferences in rectal temperatures between PRRS 94881 MLV-vaccinatedpigs and challenge controls in this study (p≧0.0528). Mean and LS Meanrectal temperatures for the negative controls remained ≦39.68° C.throughout the study.

Clinical Assessment Post-Vaccination

A summary of the percentage of piglet with at least one positiveassessment from D1 through D12 is shown below in Table 7.23.

TABLE 7.23 Percentage of Piglets With at Least One Positive ClinicalAssessments from D1-D12 Group¹ N positive % positive 95% CI Total pvalue 1 0 0 0.0 16.8 20 1.0000 2 1 5 0.1 24.9 20 3 0 0 0.0 30.8 10 NI¹Group 1 = PRRS 94881 MLV vaccine at MID, challenged; Group 2 =placebo-treated, challenged; Group 3 = placebo-treated, not challenged.NI = Not included in statistical analysis.

No piglets in either the PRRS 94881 MLV-vaccinated group or the negativecontrols had any clinical assessment findings during the vaccinationperiod D−1 through D12. Piglet 110 in challenge control group wasobserved with a sore behind the right front leg beginning on D9. Therewas no significant difference between PRRS 94881 MLV-vaccinated pigletsand challenge controls for this parameter (p=1.0000).

PRRS Serology

A summary of the frequency of piglets with positive PRRS-antibody titersis shown below in Table 7.24.

TABLE 7.24 Frequency of Piglets with Positive PRRS-Antibody Titer by DavDay Group¹ N positive % positive 95% CI Total p value 7 1 0 0 0.0 16.820 NA 2 0 0 0.0 16.8 20 3 0 0 0.0 30.8 10 NI 14 1 17 85 62.1 96.8 20<0.0001 2 0 0 0.0 16.8 20 3 0 0 0.0 30.8 10 NI 17 1 19 95 75.1 99.9 20<0.0001 2 0 0 0.0 16.8 20 3 0 0 0.0 30.8 10 NI 21 1 20 100 83.2 100.0 20  0.0012 2 11 55 31.5 76.9 20 3 0 0 0.0 30.8 10 NI 24 1 20 100 83.2100.0 20   1.0000 2 19 95 75.1 99.9 20 3 0 0 0.0 30.8 10 NI ¹Group 1 =PRRS 94881 MLV vaccine at MID, challenged; Group 2 = placebo-treated,challenged; Group 3 = placebo-treated, not challenged. NA = Notapplicable, no analysis conducted. NI = Not included in statisticalanalysis.

All piglets in all treatment groups were PRRS-antibody negative on D0and D7. By D14, 85% of the PRRS 94881 MLV-vaccinated pigs had positivePRRS antibody titers. This number increased to 95% on D17 and was 100%on both D21 and D24. No pigs in the challenge control group developedpositive PRRS-antibody titers until D21 (7 days after challengeadministration) when 55% of the pigs had positive titers. This valueincreased to 95% by D24. On D14, D17 and D21, the PRRS 94881MLV-vaccinated pigs had a significantly higher proportion of pigs withpositive PRRS antibody titers compared to the challenge control group(p≦0.0012). No pigs in the negative control group developed PRRSantibody titers during this study.

Discussion/Conclusion

To achieve the study objective, three groups were included in the studydesign on D0: a vaccine group that received 1×10^(3.82) TCID₅₀ of PRRS94881 MLV (Group 1); a challenge control group that received controlproduct (Group 2) and a negative control group (Group 3) that alsoreceived control product.

Twenty (20) healthy, PRRS susceptible and seronegative piglets wereinoculated IM with 1 ml of PRRS 94881 MLV at approximately 14 days ofage. Thirty (20 piglets—challenge control group and 10 piglets—negativecontrol group) PRRS susceptible and seronegative piglets were inoculatedIM with 1 ml of control product at approximately 14 days of age.

To determine if an onset of immunity of 2 weeks for PRRS 94881 MLV wasachieved, the vaccine group and the challenge control group werechallenged 14 days post-vaccination with a heterologous European isolateof PRRSv (isolate 205817) and evaluated post-challenge for relevantreduction in lung lesions.

Validation of the Study (Negative Control Group 3)

To ensure that source piglets were free of PRRSv and that no extraneousPRRSv exposure or cross-contamination among treatment and control groupsoccurred during the study, a negative control group (Group 3) wasincluded in the study design. Piglets in the negative control group werenegative for PRRSv (viremia; qPCR) as well as for PRRS antibodiesthroughout the study, thus validating this trial.

Validation of the Challenge Model (Challenge Control Group 2)

A challenge model that induces sufficient PRRS clinical disease isnecessary to adequately evaluate PRRS vaccine onset of immunity in alaboratory setting. Following inoculation with European PRRS isolate205817 by the method described earlier, the challenge control groupexhibited a mean rectal temperature of ≧40.50° C. on D19, D20 D23 andD24 39.68° C. on same days, negative control group), a mean ADWG of 0.15kg/day compared with a mean ADWG of 0.34 kg/day for the negative controlgroup from D14 to D24, abnormal behavior, coughing, and a median lunglesion score of 55.2% (0.00%; negative control group). These resultshighlight that severe PRRS-specific clinical disease was induced in thechallenge control group even though the challenge virus titer wasslightly lower than the targeted dose, thus validating this challengemodel as an adequate clinical laboratory tool to evaluate PRRS vaccineefficacy and more specifically, the OOI of PRRS 94881 MLV.

Determination of Two Week Onset of Immunity of PRRS 94881 MLV (Group 1)

Determination of an onset of immunity (OOI) for PRRS 94881 MLV of 2weeks post-vaccination was based upon the vaccine group exhibiting asignificant (p≦0.05) reduction in post-challenge lung lesions comparedwith the challenge control group.

Lung lesions were selected as the primary parameter for determination of2 week OOI because this parameter provides the most clinically relevantand convincing evidence of efficacy when evaluating a new vaccine withinthe PRRS respiratory challenge model in pigs. Lung lesion development isone of the hallmarks of PRRS respiratory disease in pigs. Lung lesionsare often accompanied by subsequent manifestations of secondary PRRSvdisease characteristics such as clinical signs, pyrexia, decreased ADWG,etc.

The PRRS 94881 MLV-vaccinated group exhibited a significant reduction ingross lung pathology post-challenge, as evidenced by median total lunglesion score of 27.6% in comparison to the challenge control group,which exhibited a median total lung lesion score of 55.2% (p=0.0002).Thus, an OOI of 2 weeks for PRRS 94881 MLV at dosage of 1×10^(3.82)TCID₅₀ was established based upon the primary parameter of a significantreduction for lung lesions post-challenge. This result was achieved witha vaccine dose slightly lower than the minimum immunizing dose of1×10^(4.5) TCID₅₀/dose.

Viremia post-challenge was selected as the most important secondaryparameter because it represents the level of viral replication andpersistence occurring within the host animal upon exposure. Asignificant (p≦0.05) reduction in viremia would correspond with a PRRSvaccine that induces adequate immunity to limit PRRS pathogenesis withinthe host. At 3 days post-challenge (D17), PRRS 94881 MLV-vaccinatedgroup was associated with a significant reduction in median viremia(qPCR) compared with the challenge control group (6.72 GE/mL vs. 8.18GE/mL; p≦0.0001). To further evaluate viremia post-challenge betweengroups, the quantity of the viral load over a specific duration of timepost challenge was calculated, as represented as “area under curve” orAUC. The PRRS 94881 MLV-vaccinated group had a median AUC value from D17to D24 of 49.52 GE/mL/day; while the challenge control group had amedian AUC value of 54.35 GE/ml/day. The median AUC value wassignificantly lower for the vaccine group compared with the challengecontrol group from D17 to D24 (p=0.0039). Whether viremia was examined 3days post-challenge or over the course of the post-challenge period,PRRS 94881 MLV administered 2 weeks prior to challenge with a virulentheterologous European strain of PRRS significantly (p≦0.05) reducedviremia after challenge inoculation.

In association with a reduction of PRRS viremia post-challenge, asignificant (p≦0.05) reduction in the viral load in lung tissue wouldalso be of great importance from the standpoint of PRRS vaccineimmunity. A reduction of viral load in the lung tissue maybe associatedwith reduced viral stability, replication and persistence within thehost and may secondarily lead to reduced shedding of PRRSv to otherpigs. In this study, lung tissues from PRRS 94881 MLV-vaccinated grouphad a median lung qPCR result of 7.46 log₁₀ GE/mL 10 days post challenge(D24) while the challenge control group had a median lung qPCR result of7.88 log₁₀ GE/mL. The difference between the vaccine group and thechallenge control group was significant (p=0.0101), thus furthersupporting an OOI of 2 weeks.

A marked reduction in severity and frequency of clinical signspost-challenge in piglets would also be supportive of PRRS vaccineefficacy and establishment of an OOI of 2 weeks for PRRS 94881 MLV.Abnormal respiration of sufficient severity and frequency was not notedin either group post-challenge and no differences were detected(p≧0.1394). Conversely, the severity and frequency of coughing was aboutequal between groups and no differences were detected (p≧0.0835).Differences were detected between groups for severity and frequency ofabnormal behavior (lethargy) post-challenge. Zero of 20 (0%) and 9 of 20(45%), PRRS 94881 MLV-vaccinated and challenge control piglets,respectively, exhibited abnormal behavior for at least one daypost-challenge (p=0.0012). Likewise, the PRRS 94881 MLV-vaccinated groupexhibited lower maximum abnormal clinical scores and mean abnormalclinical post-challenge compared with the challenge control group(p=0.0012). Total clinical scores (summation of respiration, behaviorand coughing scores) were significantly different between groups whenmaximum scores and mean scores from D15 to D24 were analyzed. Due to theinfluence of abnormal behavior scores on total scores, the PRRS 94881MLV-vaccinated group had a significantly lower maximum total score andlower mean total score compared with the challenge control group(p≦0.0103). The differences between groups for severity and frequency ofabnormal behavior further support an OOI of 2 weeks post-vaccination.

Pre-challenge, the PRRS 94881 MLV-vaccinated group had slightly highermean rectal temperatures compared with the challenge control group onD13 (39.77° C. vs. 39.39° C., respectively; p<0.0001) and on D14 (39.76°C. vs. 39.37° C., respectively; p<0.0001). Although significant (p≦0.05)differences were detected between groups pre-challenge, thesedifferences were not biologically relevant. Post-challenge, the only dayin which a significant (p≦0.05) difference was detected between groupsfor mean rectal temperature was on D16 (2 days post-challenge). On D16,vaccinated and challenge control groups had mean rectal temperatures of40.68° C. and 39.77° C., respectively, and difference between groups wassignificant (p<0.0001). The mean rectal temperature 4-5 days postchallenge elevated above 40° C. and remained above 40° C. until the endof the study for both groups.

The presence of significant abnormal behavior, viremia, lung pathologyand viral load in lung tissues due to PRRS in the challenge controlgroup resulted in significant (p≦0.05) differences between groups forADWG post-challenge. In this study, the vaccinated and challenge controlgroups had mean ADWG from D14 to D24 of 0.3 kg/day and 0.1 kg/day,respectively, and the difference between groups was significant(p=0.0003). A significant (p≦0.05) difference between groups for ADWGpost-challenge further supports the establishment of an OOI of 2 weekspost-vaccination.

Post-Vaccination Parameters Examined in this Study

No abnormal clinical assessments related to PRRS 94881 MLV vaccinationor control product were observed in piglets following inoculation on D0.One-challenge control piglet exhibited a sore behind the right front legbeginning on D9 which appeared not to be associated with administrationof the control product.

All piglets were PRRS ELISA serology negative on D0, thus confirmingthat all piglets met the inclusion criterion of being PRRS negative uponentry into the study. The majority of piglets receiving PRRS 94881 MLVPRRS sero-converted by D14 and all PRRS-vaccinated piglets wereseropositive by 7 days post-challenge (D21). Conversely, the challengecontrol remained seronegative until 7 days post-challenge, when thisgroup began to demonstrate PRRS seroconversion. The negative controlgroup remained PRRS seronegative throughout the entire study.

At 7 and 14 days post-vaccination, the PRRS 94881 MLV-vaccinated groupexhibited mean qPCR results of 3.17 and 3.30, log₁₀ GE/mL, respectively.These results highlight that within 2 weeks post-vaccination, a dosageof 1×10^(3.82) TCID₅₀ of PRRS 94881 MLV induced sufficient replicationof the MLV that is often required to build protective immunity alreadyat 2 weeks after vaccination. Conversely, the challenge control groupand the negative control group were negative for PRRSv viremia from D0to D14.

Conclusion

The significant (p≦0.05) reduction of the lung lesions, clinical signs,replication of the virus in the blood and lungs post-challenge as wellas the improvement of the growth performances support the establishmentof a 2 week OOI following vaccination with a single dose of PRRS 94881MLV at 1×10^(3.82) TCID₅₀/mL in piglets at approximately 14 days of age.

Example 8 Evaluation of Duration of Immunity of PRRS 94881 MLV inSusceptible Two Week Old Pigs Following Challenge with a HeterologousEuropean Isolate of PRRS at 26 Weeks Post-Vaccination

The objective of this vaccination-challenge study was to evaluate theduration of immunity (DOI) 26 weeks after the administration of thevaccine candidate Porcine Reproductive and Respiratory Syndrome,European-derived Isolate 94881, Modified Live Virus (PRRS 94881 MLV) to14±3 days of age PRRS seronegative pigs. The primary efficacy criterionto satisfy a DOI of 26 weeks post-vaccination was a significantreduction in (p≦0.05) lung lesions scores (gross or histological)post-challenge in the PRRS 94881 MLV vaccinate group (Group 1) comparedto the challenge control group (Group 2).

On Day 0 (D0), 22 pigs assigned to the vaccinate group received 1.0 mLIM of PRRS 94881 MLV (1×10^(4.27) TCID₅₀) IM (Group 1), 22 pigs assignedto the challenge control group received 1.0 mL IM of control product(product-matched placebo without PRRS 94881 MLV, Group 2) and 12 pigsassigned to the negative control group also received 1.0 mL IM ofcontrol product (Group 3). Groups 1 and 2 were challenged on D179 (Daypost-challenge {DPC} 0) with a virulent strain of European PRRSv andpigs were monitored 10 days post-challenge for clinical signs, averagedaily weight gain, and viremia. Pigs were necropsied on D189 (DPC 10)and gross and histological lung lesions, and lung viral load weredetermined.

Median gross lung lesion scores on D189 (DPC 10) were 0.1% and 13.8% forPRRS 94881 MLV-vaccinated pigs and challenge controls, respectively(p<0.0001). Median histological lung lesion scores on DPC 10 were 6.0and 19.5 for PRRS 94881 MLV-vaccinated pigs and challenge controls,respectively (p<0.0001). PRRS 94881 MLV-vaccinated pigs hadsignificantly less serum viral load at 3, 7 and 10 days post-challengecompared to challenge controls (p≦0.0001). The post-challenge area underthe curve (AUC) analysis for viremia from DPC 0 to DPC 10 and DPC 3 toDPC 10 were also significantly lower for PRRS 94881 MLV-vaccinated pigs(15.54 and 8.88 log₁₀ GE/mL per day, respectively) compared with thechallenge control group (44.77 and 36.43 log₁₀ GE/mL per day,respectively, p<0.0001). The median qPCR values for lung tissuescollected at necropsy were 3.69 and 6.25 log₁₀ GE/mL for PRRS 94881MLV-vaccinated pigs and challenge controls, respectively (p<0.0001).There were no significant differences in clinical signs post-challenge(p≧0.4878).

A significant reduction (p≦0.05) of gross and histological lung lesions,viral load in lung tissues collected at necropsy and post-challengeviremia for PRRS 94881 MLV-vaccinated pigs compared to challengecontrols supported vaccine efficacy against virulent PRRSv whenchallenged 26 weeks post-vaccination. The results of this studyestablish a 26 week duration of immunity post-vaccination in pigsvaccinated with PRRS 94881 MLV at 2 weeks of age. These results wereachieved with a vaccine dose of 1×10^(4.27) TCID₅₀/mL, which wasslightly below the minimum immunizing dose (1×10^(4.5)TCID₅₀/mL) forthis investigational veterinary product.

Objective(s)/Purpose of the Study

The objective of this vaccination-challenge study was to evaluate theduration of immunity (DOI) of Porcine Reproductive and RespiratorySyndrome, European-derived Isolate 94881, Modified Live Virus, Code19S1.U (PRRS 94881 MLV) administered to PRRS seronegative pigs, 14±3days of age against a virulent challenge with a heterologous Europeanisolate of PRRS at 26 weeks post-vaccination. The primary efficacycriterion to satisfy a DOI of 26 weeks post-vaccination was asignificant reduction (p≦0.05) in lung lesions scores (gross orhistological) post-challenge in the PRRS 94881 MLV vaccinate group(Group 1) compared to the challenge control group (Group 2)

Secondary efficacy parameters included post-vaccination andpost-challenge viremia, clinical assessments after vaccination, PRRSserology, post-challenge clinical observations, average daily weightgain (ADWG), rectal temperatures and lung PRRSv quantitation. Viremiapost-challenge was considered to be the most important secondaryparameter since it is an objective and quantifiable parameter. Rectaltemperature and clinical observations were then considered supportive inthe DOI definition process. Lastly, growth performance, serology andvirus detection in lungs were used as supportive parameters towards theprimary parameters in satisfying the study objective.

Schedule of Events

TABLE 8.1 Schedule of Events Study Day Dates Key Study Event −7  4 Feb.2010 Blood samples collected to screen for negative PRRS ELISA status −2 9 Feb. 2010 Health Exam performed −1 to 21 10 Feb. 2010- ClinicalAssessments performed daily  4 Mar. 2010 0 11 Feb. 2010 Body weightsrecorded; Blood samples collected for serology and viremia; Group 1vaccinated with IVP, Groups 2 & 3 vaccinated with CP 7 18 Feb. 2010Blood samples collected for serology and viremia 13 24 Feb. 2010 Pigsvaccinated with 1.0 mL CIRCOFLEX ® vaccine Microchip inserted SC in theleft neck of each study pig 14 25 Feb. 2010 Blood samples collected forserology and viremia 21  4 Mar. 2010 Blood samples collected forserology and viremia 22 to  5 Mar. 2010- Clinical Assessments at least 3times/week 177  7 Aug. 2010 28 11 Mar. 2010 Blood samples collected forserology and viremia 56 8 Apr. 2010 Blood samples collected for serologyand viremia 84 6 May 2010 Blood samples collected for serology andviremia 112 3 Jun. 2010 Blood samples collected for serology and viremia140  1 Jul. 2010 Blood samples collected for serology and viremia 168 29Jul. 2010 Blood samples collected for serology and viremia D178  8 Aug.2010- Daily Clinical Observations and Rectal (DPC-1) 19 Aug. 2010Temperatures to D189 (DPC 10) D179  9 Aug. 2010 Body weights collected;Blood samples (DPC 0)  collected for serology and viremia; Groups 1 and2 challenged with heterologous European PRRS isolate D182 12 Aug. 2010Blood samples collected for serology and (DPC 3)  viremia D186 16 Aug.2010 Blood samples collected for serology (DPC 7)  and viremia D188 18Aug. 2010 Body weights collected (DPC 9)  D189 19 Aug. 2010 Bloodsamples collected for (DPC 10) serology and viremia Pigs euthanized andnecropsied Lung lesions scored for pathology Lung tissues collected forvirus isolation and histopathology

Study Design

This was a blinded vaccination-challenge efficacy study conducted in 56weaned, PRRS seronegative pigs, 14±3 days of age on Day 0 (D0). Asummary of the study is provided in Table 8.2.

TABLE 8.2 Study Design Challenged on D179 (DPC 0) with 1.0 mL/ nostriland 1.0 mL IM of Euthanized Treatment PRRSv and Number on D0 205817(mean Necropsied of Pigs (14 ± 3 1 × 10^(6.27) on D189 Group on D0 daysof age) TCID₅₀/3 mL) (DPC 10) 1 22 1.0 mL IM Yes Yes of IVP (PRRS 94881MLV) 2 22 1.0 mL IM of Yes Yes Control Product (CP; Placebo matchedproduct without PRRS 94881 MLV) 3 12 1.0 mL IM of CP No Yes

Blinding Criteria

The Study Investigator and designees were blinded to the assignedtreatment groups throughout the in-life phase of the study. To maintainthis blinding, the BIVI monitor performed the randomization and anindividual who did not participate in assessments of the pigs (i.e.,clinical assessments, clinical observations or necropsies) administeredthe assigned IVP and CP treatments on D0. BIVI laboratory personnel wereblinded to the treatment each pig received while conducting theirrespective tasks.

Materials

Investigational Veterinary Product (IVP) and Control Product

TABLE 8.3 IVP Generic Product Name: Porcine Reproductive and RespiratorySyndrome, Modified Live Virus Strain: 94881 Production and BIVI-St.Joseph Production produced PRRS 94881 MLV, Formulation: Lot 390-005(Section 15.4) in accordance with Outline of Production, Code 19S1.U_and EU Dossier Part 2b. On D0, BIVI-Ames reconstituted/diluted PRRS94881 MLV vaccine Lot 390-005 with Phosphate buffered saline (PBS; Lot809-002, Section 15.5) to formulate the IVP, Lot No. N257-137 at atarget dosage of approximately 1 × 10^(4.5) TCID₅₀/mL. Manufacturer:Boehringer Ingelheim Vetmedica, Inc. 2621 North Belt Highway St. Joseph,MO 64506, USA Lot No.: 390-005, reconstituted to Lot N257-137 ExpiryDate: An expiration date of 11 Feb. 2010 was assigned to the IVP forstudy purposes only. Storage Conditions: Lyophilized vaccine: 2-8° C.Rehydrated/diluted IVP: on ice Testing: PRRS 94881 MLV, Lot 390-005 andPBS, Lot 809-002 were tested by BIVI-QC in accordance with draft Outlineof Production (Section 15.1) and as further specified in the EU dossierPart 2.F. BIVI-Ames laboratory personnel tested pre- andpost-vaccination aliquots of the WP for virus titer in accordance withthe PRRSv Titer Procedure (Section 15.1). Test Results: Lot 390-005:Results were satisfactory (Section 15.4). Lot 809-002: Results weresatisfactory (Section 15.5). IVP Lot N257-137: Mean titer of 1 ×10^(4.27) TCID₅₀/mL (Section 15.7). IVP IVP was reconstituted/dilutedfor immediate use in this study Retention: only and was not retainedbeyond the vaccination event.

TABLE 8.4 CP Generic Product Placebo Name: Formulation: BIVI-Productionproduced lyophilized placebo product containing inert material comprisedin the vaccine serial without PRRS 94881 MLV (Lot N240-191-062409,Section 15.6). On D0, BIVI-Ames reconstituted Lot N240-191-062409 withPhosphate buffered saline (PBS; Lot 809-002, Section 15.5) to formulatethe CP, Lot No. N257-134 Manufacturer: Boehringer Ingelheim Vetmedica,Inc. 2621 North Belt Highway St. Joseph, MO 64506, USA Lot Number: LotN240-191-062409, reconstituted to Lot N257-134 Expiry Date: Anexpiration date of 11 Feb. 2010 was assigned to the CP for studypurposes only. Storage Conditions: Lyophilized vaccine: 2-8° C.Rehydrated CP: 2-8° C. or on ice Testing: Lot N240-191-062409 and LotN257-134 were tested by BIVI-QC for EP sterility Test Results: LotN240-191-062409: Results were satisfactory for sterility (Section 15.6).Lot 809-002: Results were satisfactory for sterility CP was determinedto be sterile (Section 15.7). CP Retention: CP was reconstituted for usein this study only and was not retained beyond the vaccination event.

Challenge Material

TABLE 8.5 Challenge Material Name/number of PRRS isolate 205817 isolateLocation and date The European PRRS virus isolate 205817 was derivedfrom isolate of isolation incl. 190136 originally obtained from lungtissue of a newborn pig from a clinical symptoms farm with typicalreproductive signs of PRRS (abortions in sows and weakness in newbornpigs) during an outbreak in Lower Saxony, Germany, in April 2004. Theattending veterinarians submitted the lung samples to bioScreen (samplearrived on 21 Apr. 2004) for diagnostic testing. Isolate 190136 wasdirectly propagated on MA 104 cells and a pure culture challenge stockwas prepared. A pure culture of isolate 190136 was used to inoculatepigs for evaluation of its ability to reproduce PRRS-specificrespiratory disease in a controlled, laboratory trial. Challengedanimals exhibited respiratory distress and revealed evidence ofinterstitial pneumonia upon histopathological examination. PRRS virussuccessfully re-isolated from lung lesions was given the isolatedesignation of 205817. Isolate 205817 was directly propagated on MA104cells and a pure culture challenge stock was prepared for use in futureBIVI clinical trials. Formulation: Challenge virus was propagated inAK-MA104 cells and formulated to a targeted titer of approximately 1 ×10⁶ TCID₅₀/3 mL dose on D179. An adequate volume of challenge materialwas prepared. Two × 5 mL aliquots were removed from challenge materialfor assay purposes before the challenge material was transported to VRI.Lot Number: N270-179 Manufacture: Boehringer Ingelheim Vetmedica,Inc.-USA Storage conditions Bulk challenge material was stored at −70 ±10° C. Once prepared, diluted challenge material was maintained on iceuntil it was administered. Testing: BIVI-Ames laboratory personneltested pre- and post-challenge aliquots for virus titer in accordancewith the PRRSv Titer Procedure Test Results: The challenge material hada mean titer of 1 × 10^(6.27) TCID₅₀/3 mL dose Administration 1.0mL/nostril and 1.0 mL IM in the right neck route Challenge materialChallenge material was thawed/diluted for this study only and was notretention: retained beyond the challenge event.

Treatments

Dosing Justification

The IVP was administered as a 1.0 mL dose to assigned pigs to evaluateDOI of PRRS 94881 MLV at 26 weeks post-vaccination. The CP wasadministered as a 1.0 mL dose to Groups 2 and 3 as a placebo vaccine.

Dosing Regimen

IVP or CP IVP or CP was administered was administered to an assigned pigin the right neck region IM on D0 using a sterile 3.0 mL Luer-locksyringe and a sterile 20 g×1 inch (2.54 cm) needle by a person notcollecting study data. The dosing regimen is shown below in Table 8.6

TABLE 8.6 Dosing Regimen Group Number Treatment Dose/Route Study Day 122 IVP 1.0 mL IM D0 2 22 CP 1.0 mL IM D0 3 12 CP 1.0 mL IM D0

Concomitant Treatments

Due to the fact that several pigs were found dead early in the studysubsequent to bacterial infections, the Investigator and Study Monitoragreed upon the administration of the following additional concomitanttreatments to all study animals (Section 15.10):

Day 20: Mu-Se (Vitamin E/Selenium, Intervet/Schering Plough AnimalHealth, USA), 0.1 mL IM in the right ham

Day 21: EXCEDE® (Ceftiofur, Pfizer Animal Health, USA), 0.5 mL in theleft ham

Day 35: EXCEDE® (Ceftiofur, Pfizer Animal Health, USA), 1.0 mL in theright ham.

Day 42: EXCEDE® (Ceftiofur, Pfizer Animal Health, USA), 1.0 mL in theleft ham.

Day 47: BAYTRIL 100® (Enrofloxacin, Bayer Animal Health, USA), 1.5 mL SCin the left neck

Vitamin E/Selenium was administered for the prevention of mulberry heartdisease and the antibiotic treatments were administered for thetreatment/prevention of bacterial infections.

Animal Information

Details of Animal Studies

TABLE 8.7 Animal Information Source: Prairie View Farms, N5627 Hwy DD,Burlington, WI 53105 Number of 56 pigs: Arrival day: Pigs arrived at theVeterinary Resources, Inc. (VRI) Cambridge facility on D-2 (9 Feb.2010). Identification: Each animal was identified with individual doubleear tags at arrival on D-2. Each animal also had an electronic microchipinserted SC in the left neck on D13. Species: Porcine Breed: Commercialcrossbred Gender: Females or castrated males. Age range: 13 to 17 daysof age on D0 Weight 2.4 to 5.4 kg on D0 range: Ownership of BoehringerIngelheim Vetmedica, Inc. test animals: Physiological On D-2, pigsselected for assignment to the study were observed by status: the StudyInvestigator and determined to be in good health and nutritional status.Observations were recorded on the Animal Health Examination Record form.Group-Pig Group 1 (n = 22): 117, Group 2 (n = 22): 123, Group 3 (n =12): 116, Assignments 118, 119, 121, 127, 124, 125, 130, 134, 120, 126,132, 135, 128, 129, 131, 133, 137, 138, 148, 149, 145, 151, 152, 155,136, 139, 141, 142, 150, 156, 157, 158, 159, 166 and 171 144, 146, 147,153, 160, 161, 165, 167, 154, 162, 163, 164 169, 170, 172, 177 and 179and 178

Inclusion/Exclusion Criteria

All pigs enrolled in this study were PRRS ELISA negative (ELISA S/Pratio of <0.4) and were healthy at the time of vaccination (D0) asdetermined by observation.

Post-Inclusion Removal Criteria

No pigs were removed from the study. Three pigs were found dead beforechallenge administration. Further results on these three pigs arepresented in Section 12.8.

Animal Management and Housing

Pigs were housed at Veterinary Resources, Inc. (VRI) in Cambridge, Iowafor the duration of the study. Pigs were housed in multiple pens (11 or12 pigs/pen) within each room, with vaccinated (Group 1) and controlanimals (Groups 2 and 3) housed in uniform but separate rooms to ensurebiosecurity. PRRS 94881 MLV pigs were housed in Room CB8 until D78, thenin CC1 until D105, and then CC3 for the remainder of the study.Challenge control pigs were housed in Room CC2 throughout the study.Negative control pigs were housed in Room CB6 until D73 and then in CB7for the remainder of the study. Animal pens were elevated with plasticslatted flooring, with age appropriate feeders and nipple cup drinkers.Each isolation room was constructed identical to the others and all werebiohazard level 2 (BL2) compliant, hepafiltered, mechanically ventilatedwith thermostat regulated temperature control.

Treatment group isolation was necessary in this study as it is wellknown within the scientific community that PRRSv readily spreads frompig to pig via various mechanisms including aerosolization. Thisincludes avirulent live PRRS vaccines as these biological productsinclude attenuated virus particles that mimic the characteristics ofvirulent wild-type PRRS without the capability to cause disease. Propermethods were in place to ensure that biosecurity was maintained and thatvaccinated animals did not accidentally cross-contaminatenon-vaccinated, PRRSv naïve negative control animals.

Appropriate measures were taken by test facility staff to adequatelyclean and disinfect each room prior to its usage for this study.

Each room in the facility had fans and heaters to aid in sufficient aircirculation and heating. The ventilation system was separate yetidentical for each room, so air was not shared between rooms.

Feed was stored in bags, free from vermin. Feed and water were availablead libitum. Pigs were fed Lean Metrics Infant Medicated feed (PurinaMills LLC, St. Louis, Mo.) from arrival to D5, when they were switchedto Lean Metrics Senior Medicated feed (Purina Mills LLC, St. Louis,Mo.). On D64 the pigs were switched to Lean Metrics Complete 85 feed(Purina Mills LLC, St. Louis, Mo.), and on D82 they were switched toLean Metrics Complete CE85, T40 (Purina Mills LLC, St. Louis, Mo.),which they were fed for the remainder of the study. Throughout thestudy, the feeds provided were appropriate for the size, age, andcondition of the pigs according to acceptable animal husbandry practicesfor the region.

The pigs were in good health and nutritional status before initiation ofthe study as determined by the Study Investigator. During the study,select animals were observed with other conditions, including thinness,coughing, swellings, rough hair coat, depression, abscesses, and poorbody condition. The Study Investigator considered all of theseconditions to be typical of group housed growing/maturing pigs. Theseconditions were considered transient or inconsequential and were nottreated.

Assessment of Effectiveness

To assess the DOI of PRRS 94881 MLV at 26 weeks post-vaccination, thePRRS 94881 MLV and challenge control groups were challenged on D179 (DPC0) and lung lesions post-challenge were evaluated 10 days later (DPC10). A DOI of 26 weeks post-vaccination was achieved if PRRS 94881 MLVgroup had significantly decreased (p≦0.05) lung pathology (gross orhistological) post-challenge compared with the challenge control group.

The secondary efficacy parameters analyzed between the vaccine group andthe challenge control group included post-vaccination and post-challengeviremia, post-challenge clinical observations, post-challenge rectaltemperatures, post-vaccination clinical assessments, average dailyweight gain (ADWG) and PRRS serology. Viremia post-challenge wasconsidered to be the most important secondary parameter since it is anobjective and quantifiable parameter. Rectal temperature and clinicalobservations were then considered supportive in the DOI definitionprocess. Lastly, growth performance, serology and virus detection inlungs were used as supportive parameters towards the primary parametersin satisfying the study objective.

Criteria for a Valid Test

All pigs were required to be PRRS ELISA negative (ELISA S/P ratio of<0.4) at pre-purchase screening and on D0. Challenge control pigs wererequired to be negative for PRRS antibodies up to challenge and thenegative control group was required to be negative for PRRS antibodiesthroughout the study.

Primary Outcome Parameter

The primary efficacy outcome variable was lung pathology (gross andhistological lesions) at D189 (DPC 10) of the study.

Gross Lung Lesion Scores

On D189, after samples and data were collected and recorded, allremaining study pigs were euthanized following VRI SOP PRC1027 (Section15.1). Each pig was necropsied in accordance with VRI SOP PRC 1028(Section 15.1). The thoracic cavity of each pig was exposed by adesignee and the heart and lungs were removed. The Study Investigatorexamined each set of lungs, described any gross pathology and determinedthe percentage of pathology for each lung lobe. Observations and datawere recorded on the Necropsy Report Record form.

Histological Lung Lesion Scores

For each set of lungs, two samples from the Left and Right Apical lobes,the Left and Right Cardiac lobes, the Left and Right Diaphragmatic lobesand the Intermediate lobe were retained. Each lung sample wasapproximately 1 inch (2.54 cm)×1 inch (2.54 cm). For one set of lungsamples, all three samples from the left side were combined into onecontainer; while all three samples from the right side and theIntermediate lung lobe sample were combined into another container. Eachcontainer was filled with a sufficient amount of 10% formalin solution.For the other set of lung samples, all three lung samples from the leftside were combined into one WHIRLPAK®; while all three samples from theright side and the Intermediate lung lobe sample were combined intoanother WHIRLPAK®. All containers and WHIRLPAKS® were appropriatelylabeled with animal number, study number, date of collection, study day,sample type and whether the samples were from the left or right side.Lung samples in formalin were stored at room temperature while lungsamples in WHIRLPAKS® were stored on dry ice until transported toBIVI-Ames. Sample collections were recorded on the Necropsy ReportRecord form. Formalin fixed lung tissue samples and WHIRLPAK® lungsamples were transferred to BIVI-Ames. A completed Specimen DeliveryRecord form was included with each shipment.

Formalin fixed lung tissue samples were held by BIVI-Ames at roomtemperature until submitted to Iowa State University VeterinaryDiagnostic Laboratory (ISU VDL) by BIVI-Ames. Lung samples were handledand processed by ISU VDL personnel according to ISU VDL procedureswithin one week of necropsy. A single slide was generated for each pigcontaining seven sections (one each of all seven lung lobes). Each H & Eslide was identified with a unique identifier code. ISU VDL provided acomputer record containing the study number, identifier codes andassociated pig tissues.

Once daily, on the days in which the study slides were read forhistopathology, an ISU VDL pathologist (K. Schwartz) first read the EUPRRS positive and negative control slides. Afterwards, the pathologistread the H & E stained lung slides for pneumocytic hypertrophy andhyperplasia, septal infiltration with mononuclear cells, necroticdebris, intra-alveolar accumulation of inflammatory cells andperivascular accumulation of inflammatory cells. Results were recordedin an Excel spreadsheet. The lung histopathology scoring system is shownbelow in Table 8.8.

TABLE 8.8 Lung Histopathology Scoring System Pneumocytic hypertrophy andIntra-alveolar accumulation of hyperplasia inflammatory cells 0 = Notpresent 0 = Not present 1 = Mild 1 = Mild 2 = Moderate 2 = Moderate 3 =Severe 3 = Severe Septal infiltration with Perivascular accumulation ofmononuclear cells inflammatory cells 0 = Not present 0 = Not present 1 =Mild 1 = Mild 2 = Moderate 2 = Moderate 3 = Severe 3 = SevereDefinitions of scoring system applied to histological parameters (exceptnecrotic Necrotic debris debris): 0 = Not present 0 = not present: nodetectable lesions 3 = Yes present present within an area of view 1 =Mild lesions: few positive cells (1-5 cells/area) present within an areaof view 2 = Moderate lesions: multiple positive cells (>5 cells/area) atsingle location or few cells (1-5 cells/area) at multiple locationswithin an area of view. 3 = Severe lesions: multiple positive cells (>5cells/area) at multiple locations within an area of view.

Upon completion of the reading of all slides, slides were returned tothe Sponsor Representative and will be archived at Boehringer IngelheimVetmedica, Inc., St. Joseph, Mo. upon completion of the final report.

Secondary Parameters

Secondary variables included post-vaccination and post-challengeviremia, post-challenge clinical observations, post-challenge rectaltemperatures, average daily weight gain (ADWG), lung PRRSv quantitation,post-vaccination clinical assessments, and PRRS serology.

Serum PRRS qPCR

Venous whole blood was collected at pre-purchase and on Days 0, 7, 14,21, 28, 56, 84, 112, 140, 168, 179 (DPC 0), 182 (DPC 3), 186 (DPC 7),and 189 (DPC 10). Briefly, approximately 2-5 mL of blood was collectedfrom each pig into an appropriate sized serum separator tube (SST).Sample collections were recorded on the Sample Collection Record form.Blood in SSTs was allowed to clot at room temperature. Blood sampleswere delivered to BIVI-Ames on the day of collection and SpecimenDelivery Record form was completed. Blood samples were spun down byBIVI-Ames and serum was harvested, split and transferred to appropriatetubes. Each tube was labeled with the pig's ID number, the study number,the date of collection, the study day and the sample type. At BIVI-Ames,one set of serum samples was held at 2-8° C. and the other set of serumsamples was held at −70±10° C.

Clinical Observations Post-Challenge

Pigs were observed for clinical signs of disease from D178 (DPC−1) toD189 (DPC 10). Observations were conducted by the Study Investigator ordesignees and were recorded on the Clinical Observation Record form.Pigs were observed each day for respiration, behavior and cough based onthe clinical observation scoring system outlined below in Table 8.9.

TABLE 8.9 Clinical Observation Scoring System Respiration Behavior Cough0 = normal respiration 0 = normal 0 = no coughing 1 = panting/rapidrespiration 1 = mild to moderate lethargy 1 = soft or intermittent cough2 = dyspnea 2 = severely lethargic or 2 = harsh or severe, repetitive 3= dead recumbent cough 3 = dead 3 = dead

Rectal Temperatures

Rectal temperatures were collected by the Study Investigator ordesignees from D178 (DPC−1) to D189 (DPC 10). Rectal temperatures wererecorded in ° C. units on the Clinical Observation Record form.

Body Weight and Average Daily Weight Gain

Individual body weights were collected on D0, D179 (DPC 0) and D188 (DPC9). Each pig was weighed on a calibrated scale by the Study Investigatoror designees. Results were recorded in kg units on the Body WeightRecord form. Average daily weight gain was determined from the D179 (DPC0) to D188 (DPC 9).

Lung PRRS qPCR

Lung tissue samples in WHIRLPAKS® were held at −70±10° C. at BIVI-Amesuntil shipped to address listed in Section 9.3.1. A completed SpecimenDelivery Record form was included with the shipment. bioScreen testedlung samples for PRRSv RNA by qPCR (Section 15.1). Left lung tissueswere homogenized and tested. Right lung tissues and intermediate lunglobe samples were homogenized and tested. Results were reported asgenome equivalent (log₁₀ GE/mL) for left and right lung samples. Ageometric mean titer of right and left GE/mL values will be calculatedfor each pig by the statistician using SAS program.

Clinical Assessment Post-Vaccination

All pigs were observed for clinical assessments post-vaccination by theStudy Investigator or designees. Observations were conducted daily fromD−1 to D21 and then at least three times a week from D22 to D177.Observations were recorded on the Clinical Assessment Record form.

PRRS Serology

The serum samples collected at pre-purchase and on Days 0, 7, 14, 21,28, 56, 84, 112, 140, 168, 179 (DPC 0), 182 (DPC 3), 186 (DPC 7), and189 (DPC 10) and held at 2-8° C. were tested by BIVI-Ames for PRRSantibodies (Section 15.1). Results were reported as negative (ELISA S/Pratio of <0.4) or positive (ELISA S/P ratio of ≧0.4).

Adverse Events

No adverse events attributed to PRRS 94881 MLV were noted in this study.

Statistical Methods

Experimental Unit

Treatment groups were housed in separate rooms in this study to avoidtransmission of PRRSv to non-vaccinated groups. Therefore, room was theexperimental unit. However, for the purposes of analyses, possible biasdue to confounding “room” and “treatment” effects were ignored, and pigwas used as the statistical unit.

Randomization

Fifty-six (56) pigs were randomly assigned to one of three groups.Randomization was performed by the BIVI. At the time of shipment Nos.140 and 143 (challenge control group), as well as No. 168 (PRRS 94881MLV group), were culled. Number 178 was randomly selected to replace No.140, No. 177 was randomly selected to replace 143, and No. 179 wasrandomly selected to replace No. 168 from a pool of five extra pigs thatmet the inclusion criteria.

Analysis

Statistical analyses and data summaries were conducted by Dr. rer. hort.Martin Vanselow, Biometrie & Statistik, Zum Siemenshop 21, 30539Hannover, Germany, +49(0) 511 606 777 650, m.vanselow@t-online.de. Datawere analyzed assuming a completely random design structure. Thestatistical analyses were performed using SAS software release 8.2 orhigher (SAS, 2001, Cary, USA/North Carolina, SAS Institute Inc.). PRRS94881 MLV pig 179 and challenge control pigs 124 and 161 died beforechallenge and were excluded from post-challenge analyses. All tests ondifferences were designed as two-sided tests at α=5%. The statistician'sreport is presented in Section 15.9.

Gross Lung Lesion Scores

The gross lung lesion score for each pig was calculated using thefactors shown below in Table 8.10 multiplied by the % pathology for aspecific lung lobe. Calculations were conducted using SAS program.

TABLE 8.10 Factors for Calculating Gross Lung Lesion Scores Lung lobeFactor Left apical 0.05 Left cardiac 0.06 Left diaphragmatic 0.29 Rightapical 0.11 Cardiac 0.10 Right diaphragmatic 0.34 Right 0.05accessory/intermediate

Treatment groups were compared on differences using the WilcoxonMann-Whitney test.

Histological Lung Lesion Scores

Individual histological scores of the lung samples were accumulated perlobe and animal. This sum score was divided by the number of lobesexamined per animal. The results were used as single values for thecomparison between treatment groups. Treatment groups were tested ondifferences using the Wilcoxon Mann-Whitney test.

Lung PRRS qPCR

The quantitative PCR data (PRRS viral load [log₁₀ GE/mL]) from lungscollected on D189 were used for comparisons between the treatment groupsby the Wilcoxon Mann-Whitney test. The average (log₁₀ GE/mL) of the leftand right lung qPCR results were used for the evaluation. Prior to thecalculations the analytical result ‘not detected’ was replaced by log₁₀GE/mL of 0.0 and ‘positive’ was replaced by 3.0.

Frequency tables of positive qPCR results were generated. Differencesbetween treatment groups were tested by Fisher's exact test.

Serum PRRS qPCR

The viremia data were evaluated separately for each day ofinvestigation. Additionally, for viral load the areas under theindividual response curves between D179 and D189 (AUC0-10) and betweenD182 and D189 (AUC3-10) were analyzed.

The quantitative PCR data (PRRS viral load [log₁₀ GE/mL]) were used forcomparisons between the treatment groups by the Wilcoxon Mann-Whitneytest. Prior to the calculations the analytical result ‘not detected’ wasreplaced by a log₁₀ GE/mL value of 0.0 and ‘positive’ was replaced by3.0. The treatment groups were tested on differences using the WilcoxonMann-Whitney test.

Frequency tables of positive qPCR results were generated. Differencesbetween treatment groups were tested by Fisher's exact test.

Clinical Observations Post-Challenge

Frequency tables of animals with at least one positive finding betweenD180 and D189 were generated. Total scores were the summation ofrespiration score+behavior score+cough score. Calculations wereconducted using SAS program. Differences between treatment groups weretested by Fisher's exact test.

The maximum scores and the mean scores per animal from D180 to D189 forrespiration, behavior, coughing and for all three added together (total)were used for the statistical evaluation. Differences between treatmentgroups were tested by the Wilcoxon Mann-Whitney test.

Body Weight and Average Daily Weight Gain

Individual daily weight gains were calculated for the time periodbetween D179 to D188. For each day of investigation and for the timeperiod descriptive statistics were calculated. Differences betweentreatment groups were tested using analysis of variance and subsequentt-tests. Least squares means of the groups and differences between leastsquares means with 95% confidence intervals were calculated from theanalysis of variance.

Rectal Temperatures

Differences between treatment groups with respect to the originaltemperature data were tested using analysis of variance and subsequentt-tests. Least squares means of the groups and differences between leastsquares means with 95% confidence intervals were calculated from theanalysis of variance.

Clinical Assessment Post-Vaccination

Frequency tables of animals with at least one positive finding betweenD1 and D21 were generated. Differences between treatment groups weretested by Fisher's exact test.

PRRS Serology

Frequency tables of positive ELISA results were generated for each timepoint. Differences between treatment groups were tested by Fisher'sexact test.

Results

Gross Lung Lesion Scores

Median gross lung lesion scores on D189 (DPC 10) were 0.1% and 13.8% forthe PRRS 94881 MLV-vaccinated group and challenge controls,respectively. The median gross lung lesion score for PRRS-vaccinatedpigs was significantly lower than the median gross lung lesion score forthe challenge controls (p<0.0001). The median gross lung lesion scorefor the negative control group was 0.0%.

Number 123 (challenge control group) could not be scored for lunglesions on D189 due to diffuse pleuritis and adhesions. Moraxellaosloensis, Staphylococcus warneri, Staphyloccous hyicus and Pseudomonasspecies were isolated from this pig's lung tissues post necropsy.

A summary of group gross lung lesion scores and the associated p-valueis shown below in Table 8.11.

TABLE 8.11 Summary of Group Gross Lung Lesion Scores (%) on D189 Group¹N³ Min. Max. Median 95% CI Q Range Mean p value 1 21 0.00 12.40 0.0600.050 0.550 0.400 1.099 <0.0001 2 19² 0.06 69.20 13.800 2.690 22.65020.850 15.842 3 12 0.00 0.59 0.000 0.000 0.110 0.085 0.093 NI ¹Group 1 =PRRS 94881 MLV vaccine; Group 2 = Challenge control group; Group 3 =Negative control group. ²No. 123 was not scored due to diffuse pleuritisand adhesions due to bacterial infections. ³One PRRS 94881 MLV pig andtwo challenge control pigs died pre-challenge and were not included inanalysis. NI = Not included in statistical analysis

Histological Lung Lesion Scores

Median histological lung lesion scores were 6.0 and 19.5 for the PRRS94881 MLV-vaccinated group and challenge controls, respectively. Themedian histological lung lesion score for the PRRS-vaccinated group wassignificantly lower than the median histological lung lesion score forchallenge controls (p<0.0001). The median histological lung lesion scorefor the negative control group was 9.0.

A summary of the group histological lung lesion scores and theassociated p-value is shown below in Table 8.12.

TABLE 8.12 Summary of Group Histological Lung Lesion Scores Group¹ N³Min. Max. Median 95% CI Q Range Mean p value 1 21 2 20 6.0 3.0 8.0 5.06.6 <0.0001 2 20 8 47 19.5 15.0 23.0 10.0 20.2 3 12 0 15 9.0 7.0 14.06.5 9.1 NI ¹Group 1 = PRRS 94881 MLV vaccine; Group 2 = Challengecontrol group; Group 3 = Negative control group. ²One PRRS 94881 MLV pigand two challenge control pigs died pre-challenge and were not includedin analysis. NI = Not included in statistical analysis

Lung PRRS qPCR

Median qPCR lung values from lung tissues were 3.69 and 6.25 log₁₀ GE/mLfor PRRS 94881 MLV-vaccinated pigs and challenge controls, respectively.The median qPCR value for PRRS 94881 MLV-vaccinated pigs wassignificantly lower than the median qPCR value for challenge controls(p<0.0001). No PRRSv RNA was detected in lung samples of any negativecontrol pigs.

A summary of group lung qPCR values and test result (p value) is belowin Table 8.13.

TABLE 8.13 Summary of Group Lung qPCR (mean log₁₀ GE/mL) Values Group¹N² Min. Max. Median 95% CI Q Range Mean p value 1 21 0.00 6.83 3.69 1.505.21 3.18 3.36 <0.0001 2 20 4.80 7.40 6.25 5.62 6.68 1.26 6.22 3 12 0.000.00 0.00 0.00 0.00 0.00 0.00 NI ¹Group 1 = PRRS 94881 MLV vaccine;Group 2 = Challenge control group; Group 3 = Negative control group.²One PRRS 94881 MLV pig and two challenge control pigs diedpre-challenge and were not included in analysis. NI = Not included instatistical analysis

PRRSv RNA was detected in lung tissues of 90% and 100% of PRRS 94881MLV-vaccinated pigs and challenge control pigs, respectively. There wasno statistical difference between the vaccinated group and challengecontrols (p=0.4878).

A summary of group frequency of PRRS qPCR positive lung tissues frompigs at necropsy is shown below in Table 8.14.

TABLE 8.14 Group Frequency of PRRSv qPCR Positive Lung Tissues N % Pos-Total Group¹ Positive itive 95% CI Number² p value 1 19 90 69.6 98.8 210.4878 2 20 100 83.2 100.0 20 3 0 0 0.0 26.5 12 NI ¹Group 1 = PRRS 94881MLV vaccine; Group 2 = Challenge control group; Group 3 = Negativecontrol group. ²One PRRS 94881 MLV pig and two challenge control pigsdied pre-challenge and were not included in analysis. NI = Not includedin statistical analysis

Serum PRRS qPCR

PRRSv RNA was not detected in the serum of any pigs on D0.Post-vaccination, PRRS 94881 MLV-vaccinated pigs had median values of3.00, 0, 0, 3.00, 0, 0, 0, 0 and 0 log₁₀ GE/mL on D7, D14, D21, D28,D56, D84, D112, D140 and D168 respectively. The values weresignificantly higher than challenge controls on D7, D14, D21 and D28(p≦0.0013), as challenge controls did not have any PRRSv RNA detecteduntil D182 (DPC 3).

PRRSv RNA was not detected in the serum of any pigs on D179 (DPC 0).Post-challenge, PRRS 94881 MLV-vaccinated pigs had median values of4.44, 0 and 0 log₁₀ GE/mL on D182 (DPC 3), D186 (DPC 7), and D189 (DPC10), respectively, compared with 5.88, 5.30 and 4.24 log₁₀ GE/mL forchallenge controls on the same days. Median values for the challengecontrols were higher than the PRRS 94881 MLV group on all post-challengedays (p≦0.0001).

No PRRSv RNA was detected in serum from any negative control pig duringthis study.

The median AUC values for PRRS 94881 MLV-vaccinated pigs were 15.54 and8.88 log₁₀ GE/mL per day from DPC 0 to DPC 10 and from DPC 3 to DPC 10,respectively. In contrast, the median AUC values for challenge controlswere 44.77 and 36.43 log₁₀ GE/mL per day from DPC 0 to DPC 10 and fromDPC 3 to DPC 10, respectively. Median values for the PRRS MLV group weresignificantly lower than median values for the challenge controls forboth periods (p<0.0001).

Summaries of serum PRRS qPCR data are shown below in Tables 8.15 and8.16.

TABLE 8.15 Summary of Serum PRRS qPCR Results (log₁₀ GE/mL) from D 0 toD 168 Day Group¹ N Min. Max. Median 95% CI QRange Mean p value 0 1 220.00 0.00 0.00 0.00 0.00 0.00 0.00 1.0000 2 22 0.00 0.00 0.00 0.00 0.000.00 0.00 3 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI 7 1 21 3.00 4.633.00 3.00 3.00 0.00 3.23 <0.0001  2 22 0.00 0.00 0.00 0.00 0.00 0.000.00 3 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI 14 1 21 0.00 3.00 0.000.00 3.00 3.00 1.43 0.0005 2 21 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3 120.00 0.00 0.00 0.00 0.00 0.00 0.00 NI 21 1 21 0.00 3.00 0.00 0.00 3.003.00 1.29 0.0013 2 20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3 12 0.00 0.000.00 0.00 0.00 0.00 0.00 NI 28 1 21 0.00 3.88 3.00 0.00 3.00 3.00 1.93<0.0001  2 20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3 12 0.00 0.00 0.000.00 0.00 0.00 0.00 NI 56 1 21 0.00 3.00 0.00 0.00 0.00 0.00 0.57 0.10692 20 0.00 0.00 0.00 0.00 0.00 0.00 0.00 3 12 0.00 0.00 0.00 0.00 0.000.00 0.00 NI 84 1 21 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.0000 2 20 0.000.00 0.00 0.00 0.00 0.00 0.00 3 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI112 1 21 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.0000 2 20 0.00 0.00 0.000.00 0.00 0.00 0.00 3 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI 140 1 210.00 0.00 0.00 0.00 0.00 0.00 0.00 1.0000 2 20 0.00 0.00 0.00 0.00 0.000.00 0.00 3 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI 168 1 21 0.00 0.000.00 0.00 0.00 0.00 0.00 1.0000 2 20 0.00 0.00 0.00 0.00 0.00 0.00 0.003 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI ¹Group 1 = PRRS 94881 MLVvaccine; Group 2 = Challenge control group; Group 3 = Negative controlgroup. NI = Not included in statistical analysis

TABLE 8.16 Summary of Serum PRRS qPCR Results (log₁₀ GE/mL) from D 179to D 189 Day Group¹ N Min. Max. Median 95% CI QRange Mean p value 179 121 0.00 0.00 0.00 0.00 0.00 0.00 0.00  1.0000 (DPC 2 20 0.00 0.00 0.000.00 0.00 0.00 0.00 0) 3 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI 182 121 3.00 5.58 4.44 3.93 5.28 1.51 4.42 <0.0001 (DPC 2 20 5.09 6.33 5.885.75 6.00 0.32 5.81 3) 3 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI 186 121 0.00 3.74 0.00 0.00 0.00 0.00 0.61 <0.0001 (DPC 2 20 3.66 6.57 5.304.86 5.69 1.08 5.30 7) 3 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI 189 121 0.00 0.00 0.00 0.00 0.00 0.00 0.00 <0.0001 (DPC 2 20 0.00 5.88 4.243.71 4.42 1.18 3.97 10) 3 12 0.00 0.00 0.00 0.00 0.00 0.00 0.00 NI AUC 121 10.50 31.22 15.54 13.76 19.53 5.95 17.61 <0.0001 DPC 2 20 36.86 52.1644.77 43.23 48.03 6.24 44.84 0-10 3 12 0.00 0.00 0.00 0.00 0.00 0.000.00 NI AUC 1 21 6.00 23.45 8.88 7.86 11.16 3.40 10.97 <0.0001 DPC 3- 220 27.77 43.02 36.43 34.60 38.53 5.23 36.12 DPC 10 3 12 0.00 0.00 0.000.00 0.00 0.00 0.00 NI ¹Group 1 = PRRS 94881 MLV vaccine; Group 2 =Challenge control group; Group 3 = Negative control group. NI = Notincluded in statistical analysis. AUC = Area under the curve; log₁₀GE/mL per day

Post-vaccination, the PRRS 94881 MLV group had significantly higherproportions of qPCR positive pigs on D7, D14, D21 and D28 compared withthe challenge control group. (p≦0.0013). No significant difference wasdetected between groups on D56 for the proportion of qPCR positive pigs(p=0.1069).

On D182 (DPC 3), 100% of pigs in the PRRS 94881 MLV and challengecontrol groups were qPCR positive (no test conducted). On D186 (DPC 7)and D189 (DPC 10), the PRRS MLV group had significantly lower proportionof qPCR positive pigs compared with the challenge control group(<0.0001).

Summaries of group proportions of qPCR positive data are shown below inTables 8.17 and 8.18.

TABLE 8.17 Summary of Group Proportion of Serum qPCR Positive ResultsPost-Vaccination N % Pos- Total Day Group ¹ Positive itive 95% CI Numberp value 0 1 0 0 0.0 15.4 22 n.a. 2 0 0 0.0 15.4 22 3 0 0 0.0 26.5 12 NI7 1 21 100 83.9 100.0 21 <0.0001  2 0 0 0.0 15.4 22 3 0 0 0.0 26.5 12 NI14 1 10 48 25.7 70.2 21 0.0005 2 0 0 0.0 16.1 21 3 0 0 0.0 26.5 12 NI 211 9 43 21.8 66.0 21 0.0013 2 0 0 0.0 16.8 20 3 0 0 0.0 26.5 12 NI 28 113 62 38.4 81.9 21 <0.0001  2 0 0 0.0 16.8 20 3 0 0 0.0 26.5 12 NI 56 14 19 5.4 41.9 21 0.1069 2 0 0 0.0 16.8 20 3 0 0 0.0 26.5 12 NI 84 1 0 00.0 16.1 21 n.a. 2 0 0 0.0 16.8 20 3 0 0 0.0 26.5 12 NI 112 1 0 0 0.016.1 21 n.a. 2 0 0 0.0 16.8 20 3 0 0 0.0 26.5 12 NI 140 1 0 0 0.0 16.121 n.a. 2 0 0 0.0 16.8 20 3 0 0 0.0 26.5 12 NI 168 1 0 0 0.0 16.1 21n.a. 2 0 0 0.0 16.8 20 3 0 0 0.0 26.5 12 NI ¹ Group 1 = PRRS 94881 MLVvaccine; Group 2 = Challenge control group; Group 3 = Negative controlgroup, n.a. = no test conducted; NI = Not included in statisticalanalysis

TABLE 8.18 Summary of Group Proportion of Serum qPCR Positive ResultsPost-Challenge N % Pos- Total Day Group ¹ Positive itive 95% CI Number pvalue 179 1 0 0 0.0 16.1 21 n.a. (DPC 2 0 0 0.0 16.8 20 0) 3 0 0 0.026.5 12 NI 182 1 21 100 83.9 100.0 21 n.a. (DPC 2 20 100 83.2 100.0 203) 3 0 0 0.0 26.5 12 NI 186 1 4 19 5.4 41.9 21 <0.0001 (DPC 2 20 10083.2 100.0 20 7) 3 0 0 0.0 26.5 12 NI 189 1 0 0 0.0 16.1 21 <0.0001 (DPC2 19 95 75.1 99.9 20 10) 3 0 0 0.0 26.5 12 NI ¹ Group 1 = PRRS 94881 MLVvaccine; Group 2 = Challenge control group; Group 3 = Negative controlgroup, n.a. = no test conducted; NI = Not included in statisticalanalysis

Clinical Observations Post-Challenge

Abnormal respiration was not observed in any PRRS 94881 MLV-vaccinatedpigs after challenge, compared with one—challenge control pig (No. 149)which demonstrated a score of “1” on D185 (DPC 6). No difference wasdetected between groups for the percentage of pigs that demonstratedabnormal respiration for at least one day post-challenge (p=0.4878).

Abnormal behavior and coughing were not observed post-challenge in anyPRRS 94881 MLV-vaccinated pigs or in challenge control pigs.

The percentages of pigs with total clinical scores >0 for at least oneday post-challenge were 0% and 5% for the PRRS 94881 MLV-vaccinatedgroup and the challenge control group, respectively. These values werenot significantly different (p=0.4878).

No clinical signs were observed in the negative control group from D179to D189.

A summary of group frequencies of pigs with at least one positiveclinical observation score during the post-challenge period is shownbelow in Table 8.19.

TABLE 8.19 Summary of Group Frequencies of Pigs with at least OnePositive Clinical Observation Score Post-challenge N % Pos- TotalParameter Group¹ Positive itive 95% CI Number² p value Respira- 1 0 00.0 16.1 21 0.4878 tion 2 1 5 0.1 24.9 20 3 0 0 0.0 26.5 12 NI Behav- 10 0 0.0 16.1 21 NA ior 2 0 0 0.0 16.8 20 3 0 0 0.0 26.5 12 NI Cough- 1 00 0.0 16.1 21 NA ing 2 0 0 0.0 16.8 20 3 0 0 0.0 26.5 12 NI Total 1 0 00.0 16.1 21 0.4878 2 1 5 0.1 24.9 20 3 0 0 0.0 26.5 12 NI ¹Group 1 =PRRS 94881 MLV vaccine; Group 2 = Challenge control group; Group 3 =Negative control group. ²One PRRS 94881 MLV pig and two challengecontrol pigs died pre-challenge and were not included in analysis. NI =Not included in statistical analysis; NA = test not applicable due tolack of variability

There was no difference between groups for maximum respiration scores ormaximum total scores post-challenge (p=0.4878).

A summary of the group maximum clinical observation scores for thepost-challenge period (DPC 1 through DPC 10) is shown below in Table8.20.

TABLE 8.20 Summary of Group Post-Challenge Maximum Clinical ScoresParameter Group¹ N² Min. Max. Median 95% CI QRange Mean p value Respira-1 21 0 0 0 0 0 0 0.0 0.4878 tion 2 20 0 1 0 0 0 0 0.1 3 12 0 0 0 0 0 00.0 NI Behav- 1 21 0 0 0 0 0 0 0.0 1.0000 ior 2 20 0 0 0 0 0 0 0.0 3 120 0 0 0 0 0 0.0 NI Cough- 1 21 0 0 0 0 0 0 0.0 1.0000 ing 2 20 0 0 0 0 00 0.0 3 12 0 0 0 0 0 0 0.0 NI Total 1 21 0 0 0 0 0 0 0.0 0.4878 2 20 0 10 0 0 0 0.1 3 12 0 0 0 0 0 0 0.0 NI ¹Group 1 = PRRS 94881 MLV vaccine;Group 2 = Challenge control group; Group 3 = Negative control group.²One PRRS 94881 MLV pig and two challenge control pigs diedpre-challenge and were not included in analysis. NI = Not included instatistical analysis

Mean clinical observation scores followed a pattern similar to thepercentage of pigs with positive clinical scores. There were nosignificant differences between the PRRS 94881 MLV-vaccinated group andthe challenge control group (p≧0.4878).

A summary of the group mean clinical observation scores for thepost-challenge period (DPC 1 through DPC 10) is shown below in Table8.21.

TABLE 8.21 Summary of Group Post-Challenge Mean Clinical ScoresParameter Group¹ N² Min. Max. Median 95% CI QRange Mean p value Respira-1 21 0.0 0.0 0.00 0.00 0.00 0.00 0.00 0.4878 tion 2 20 0.0 0.1 0.00 0.000.00 0.00 0.01 3 12 0.0 0.0 0.00 0.00 0.00 0.00 0.00 NI Behav- 1 21 0.00.0 0.00 0.00 0.00 0.00 0.00 1.0000 ior 2 20 0.0 0.0 0.00 0.00 0.00 0.000.00 3 12 0.0 0.0 0.00 0.00 0.00 0.00 0.00 NI Cough- 1 21 0.0 0.0 0.000.00 0.00 0.00 0.00 1.0000 ing 2 20 0.0 0.0 0.00 0.00 0.00 0.00 0.00 312 0.0 0.0 0.00 0.00 0.00 0.00 0.00 NI Total 1 21 0.0 0.0 0.00 0.00 0.000.00 0.00 0.4878 2 20 0.0 0.1 0.00 0.00 0.00 0.00 0.01 3 12 0.0 0.0 0.000.00 0.00 0.00 0.00 NI ¹Group 1 = PRRS 94881 MLV vaccine; Group 2 =Challenge control group; Group 3 = Negative control group. ²One PRRS94881 MLV pig and two challenge control pigs died pre-challenge and werenot included in analysis. NI = Not included in statistical analysis

Body Weight and Average Daily Weight Gain

The difference between groups was not significant (p=0.2389). On D179(DPC 0), mean and LS Mean body weights were 134.6 and 128.2 kg for thePRRS 94881 MLV group and the challenge control group, respectively. Thedifference was not significantly different (p=0.1090). On D188 (DPC 9),the mean and LS Mean body weights were 138.3 and 130.3 kg for the PRRS94881 MLV group and the challenge control group, respectively. The bodyweight for the vaccinated group was significantly higher than thechallenge control group on D188 (p=0.0455).

LS Mean ADWGs for the challenge period (DPC 0 through DPC 9) were 0.4and 0.2 kg/d for the PRRS 94881 MLV group and the challenge controlgroup, respectively. These values were not significantly different(p=0.1041).

Negative control pigs had mean body weights of 2.7, 117.2 and 120.0 kgon D0, D179 and D188, respectively. The ADWG for the negative controlgroup from D179 to D188 was 0.5 kg/d.

A summary of the group mean body weights on D0, D179 (DPC 0) and

D188 (DPC 9) and ADWG from DPC 0 to DPC 9 are shown below in Table 8.22.A summary of LS Mean and statistical analysis of body weights and ADWGfor the PRRS 94881 MLV group and the challenge control group is shownbelow in Table 8.23.

TABLE 8.22 Summary of Group Body Weight and Average Daily Weight Gain(kg and kg/d) Day(s) Group¹ N Min. Max. Median Mean SD 0 Body 1 22 2.85.4 4.00 3.96 0.730 Weights 2 22 2.4 4.8 3.75 3.72 0.547 3 12 2.7 4.53.60 3.71 0.552 D 179 1 21 108.5 155.0 136.60 134.57 12.737 Body 2 20103.3 152.6 130.10 128.15 12.288 Weights 3 12 117.2 156.5 133.05 134.6110.900 (DPC 0) D 188 1 21 112.2 157.8 141.50 138.28 12.879 Body 2 20109.4 150.9 131.90 130.27 11.896 Weights 3 12 120.0 162.5 136.60 139.1111.922 (DPC 9) ADWG 1 21 −0.422 0.956 0.4111 0.4124 0.31653 DPC 0 to 220 −0.589 0.844 0.2889 0.2350 0.36530 DPC 9 3 12 −1.600 2.656 0.51110.5000 0.92391 ¹Group 1 = PRRS 94881 MLV vaccine; Group 2 = Challengecontrol group; Group 3 = Negative control group

TABLE 8.23 Summary of Group LS Mean Body Weight and Daily Gain (kg andkg/d) Day(s) Group¹ LS Mean 95% confidence interval p value 0 Body 13.96 3.674 4.241 0.2389 Weights 2 3.72 3.446 4.000 Diff. 1 − 2 0.23−0.162 0.631 D179 1 134.57 129.040 140.093 0.1090 Body 2 128.15 122.487133.813 Weights Diff. 1 − 2 6.42 −1.496 14.329 (DPC 0) D188 1 138.28132.800 143.756 0.0455 Body 2 130.27 124.652 135.878 Weights Diff. 1 − 28.01 0.170 15.856 (DPC 9) ADWG 1 0.4124 0.26180 0.56297 0.1041 DPC 0 to2 0.2350 0.08070 0.38930 DPC 9 Diff. 1 − 2 0.1774 −0.03822 0.39299¹Group 1 = PRRS 94881 MLV vaccine; Group 2 = Challenge control group;Group 3 = Negative control group

Rectal Temperatures

Mean rectal temperature for the PRRS 94881 MLV group was 39.3° C. on theday of challenge (D179), and means ranged from 39.1° C. (D189, DPC 10)to 39.8° C. (D181, DPC 2) after challenge. Mean rectal temperature forthe challenge control group was 39.1° C. on the day of challenge, andmeans ranged from 39.1° C. (D183, DPC 4) to 39.9° C. (D182, DPC 3) afterchallenge. Mean rectal temperatures for negative control group remained≦39.3° C. throughout the same time period.

A summary of group rectal temperatures is shown below in Table 8.24.

TABLE 8.24 Summary of Group Rectal Temperature (° C.) Days D 179 (DPC 0)through D 189 (DPC 10) Day Group¹ N² Min. Max. Median Mean SD D 179 1 2138.5 40.0 39.40 39.33 0.360 (DPC 2 20 38.6 40.0 39.00 39.07 0.380 0) 312 38.8 39.7 39.30 39.27 0.257 D 180 1 21 38.7 40.4 39.40 39.46 0.370(DPC 2 20 38.9 40.9 39.60 39.61 0.527 1) 3 12 38.8 39.5 39.00 39.090.227 D 181 1 21 39.0 41.0 39.80 39.82 0.473 (DPC 2 20 38.6 40.5 39.3539.42 0.487 2) 3 12 38.8 39.2 38.80 38.90 0.141 D 182 1 21 38.5 40.639.50 39.52 0.542 (DPC 2 20 39.0 41.1 40.05 39.86 0.588 3) 3 12 38.739.4 39.00 39.05 0.254 D 183 1 21 38.9 40.8 39.50 39.52 0.411 (DPC 2 2038.4 40.3 39.00 39.08 0.508 4) 3 11 38.8 39.4 39.10 39.08 0.209 D 184 121 39.0 40.3 39.70 39.72 0.360 (DPC 2 20 38.7 39.7 39.10 39.15 0.302 5)3 12 38.8 39.5 39.10 39.10 0.191 D 185 1 21 39.1 40.5 39.60 39.66 0.376(DPC 2 20 38.9 40.9 39.25 39.48 0.546 6) 3 12 38.4 39.4 38.85 38.880.313 D 186 1 21 38.1 40.4 39.20 39.22 0.413 (DPC 2 20 38.6 40.4 39.3539.39 0.479 7) 3 12 38.5 39.6 38.90 38.98 0.328 D 187 1 21 38.8 39.939.20 39.23 0.290 (DPC 2 20 38.8 40.8 39.45 39.58 0.573 8) 3 12 38.539.5 38.85 38.93 0.296 D 188 1 21 38.8 39.9 39.10 39.17 0.288 (DPC 2 2038.3 40.5 39.00 39.20 0.598 9) 3 12 38.4 39.1 38.85 38.85 0.173 D 189 121 38.7 39.7 39.00 39.06 0.256 (DPC 2 20 39.0 40.8 39.50 39.51 0.408 10)3 12 38.6 39.3 39.05 39.00 0.226 ¹Group 1 = PRRS 94881 MLV vaccine;Group 2 = Challenge control group; Group 3 = Negative control group.²One PRRS 94881 MLV pig and two challenge control pigs diedpre-challenge and were not included in analysis

Least square Mean rectal temperatures were significantly higher for PRRS94881 MLV-vaccinated pigs compared to challenge controls on DPC 0(p=0.0281), DPC 2 (p=0.0095), DPC 4 (p=0.0034) and DPC 5 (p<0.0001).Least square Mean rectal temperatures were significantly lower for PRRS94881 MLV-vaccinated pigs compared to challenge controls on DPC 8(p=0.0183) and on DPC 10 (p=0.0001). No significant differences weredetected between groups for the remaining days post-challenge(p≧0.0642). A summary of group LS Mean and statistical analysis ofrectal temperature is shown below in Table 8.25.

TABLE 8.25 Summary of Group LS Mean Rectal Temperature (° C.) D179 (DPC0) through D189 (DPC 10) Day Group¹ LSMean 95 % confidence interval pvalue D179 1 39.33 39.170 39.496 0.0281 (DPC 0) 2 39.07 38.903 39.237Diff. 1 − 2 0.26 0.030 0.497 D180 1 39.46 39.257 39.657 0.3025 (DPC 1) 239.61 39.400 39.810 Diff. 1 − 2 −0.15 −0.434 0.138 D181 1 39.82 39.61240.036 0.0095 (DPC 2) 2 39.42 39.198 39.632 Diff. 1 − 2 0.41 0.105 0.712D182 1 39.52 39.274 39.773 0.0642 (DPC 3) 2 39.86 39.604 40.116 Diff. 1− 2 −0.34 −0.693 0.021 D183 1 39.52 39.320 39.727 0.0034 (DPC 4) 2 39.0838.867 39.283 Diff. 1 − 2 0.45 0.158 0.740 D184 1 39.72 39.572 39.866<0.0001   (DPC 5) 2 39.15 38.999 39.301 Diff. 1 − 2 0.57 0.359 0.779D185 1 39.66 39.457 39.869 0.2164 (DPC 6) 2 39.48 39.269 39.691 Diff. 1− 2 0.18 −0.112 0.478 D186 1 39.22 39.027 39.421 0.2408 (DPC 7) 2 39.3939.188 39.592 Diff. 1 − 2 −0.17 −0.448 0.116 D187 1 39.23 39.034 39.4320.0183 (DPC 8) 2 39.58 39.376 39.784 Diff. 1 − 2 −0.35 −0.631 −0.062D188 1 39.17 38.966 39.377 0.8454 (DPC 9) 2 39.20 38.989 39.411 Diff. 1− 2 −0.03 −0.323 0.266 D189 1 39.06 38.908 39.207 0.0001 (DPC 2 39.5139.352 39.658 10) Diff. 1 − 2 −0.45 −0.662 −0.234 ¹Group 1 = PRRS 94881MLV vaccine; Group 2 = Challenge control group; Group 3 = Negativecontrol group

Three of 21 (14%) PRRS 94881 MLV vaccinated pigs and 5 of 20 (25%)challenge controls had a rectal temperature ≧40.5° C. for at least oneday post-challenge. No difference was detected between groups for theproportion of pigs that exhibited a rectal temperature ≧40.5° C. for atleast one day post-challenge (p=0.4537). A summary of group proportionof pigs with pyrexia 40.5° C.) for at least one day post-challenge isshown below in Table 8.26.

TABLE 8.26 Summary of Group Proportion of Pyrexia (≧40.5° C.) for atLeast One Day Post-Challenge N % Pos- Total Day Group¹ Positive itive95% CI Number² p value D 180 1 3 14 3.0 36.3 21 0.4537 (DPC 1) 2 5 258.7 49.1 20 to D 189 3 0 0 0.0 26.5 12 NI (DPC 10) ¹Group 1 = PRRS 94881MLV vaccine; Group 2 = Challenge control group; Group 3 = Negativecontrol group. ²One PRRS 94881 MLV pig and two challenge control pigsdied pre-challenge and were not included in analysis. NI = Not includedin statistical analysis

Clinical Assessment Post-Vaccination

Four of 22 (18%) PRRS 94881 MLV pigs, 8 of 22 (36%) challenge controlpigs and 2 of 12 (17%) negative control pigs exhibited an abnormalclinical assessment for at least one day from D1 to D21. There was nosignificant difference between groups for this parameter (p=0.3102).

A summary of group percentage of pigs with at least one abnormalclinical assessment from D1 through D21 is shown below in Table 8.27.

TABLE 8.27 Summary of Group Percentage of Pigs with At Least OneAbnormal Clinical Assessments from D 1-D 21 Total Group¹ N Positive %Positive 95% CI Number p value 1 4 18 5.2 40.3 22 0.3102 2 8 36 17.259.3 22 3 2 17 2.1 48.4 12 NI ¹Group 1 = PRRS 94881 MLV vaccine; Group 2= Challenge control group; Group 3 = Negative control group. NI = Notincluded in statistical analysis

Overall, 7 PRRS 94881 MLV pigs exhibited an abnormal clinical assessmentfor at least one day between D1 to D177:

Pig 121 exhibited belly swelling from D61 to D146, a swollen sheath fromD147 to D167, belly swelling on D168, a swollen sheath from D169-172 andbelly swelling from 173 to 177. Pig 141 was thin from D4-D10, wasdepressed from D4-D6 and rough hair coat on D5. Pig 144 exhibitedcoughing on D26. Pig 146 exhibited swelling on the sternum on D82. Pig147 was weak on legs from D84-D86 and was shaking on D84. Pig 154 wasthin from D4-D6. Pig 179 was thin from D2-D5, exhibited rough hair coaton D5 and was found dead on D6. Thirteen pigs in the challenge controlgroup exhibited an abnormal clinical assessment for at least one dayfrom D1 to D177: Pig 124 exhibited shaking and tremors on D20 and wasfound dead on D21. Pig 134 exhibited a swollen sheath from D46-D68,belly swelling from D69-D143, an umbilical hernia on D144, and bellyswelling from D145 to D177. Pig 137 exhibited a swollen sheath fromD108-D143. Pig 138 exhibited a swollen sheath from D115-D143. Pig 148exhibited lameness or a swollen leg from D16-20 and coughing on D35. Pig149 was thin from D5-D9 and on D12, and exhibited rough hair coat fromD12-D15. Pig 150 was thin from D4-D9 and on D13, exhibited poor bodycondition from D10-D12, and was depressed on D11. Pig 161 was thin fromD4-D9, exhibited rough hair coat and central nervous system signs on D9and was found dead on D10. Pig 167 exhibited a swollen sheath fromD117-D143. Pig 170 was thin from D4-D7 and exhibited depression on D7.Pig 172 exhibited a sore or a swollen dew claw from D120-D143. Pig 177exhibited depression on D19 and swelling on the neck from D156-D159. Pig178 exhibited depression on D5, from D17-20, and from D28-D36, was lameand/or swollen leg from D15-D47, was thin from D16-D18, and was stiff onlegs from D39-D47. Six pigs in the negative control exhibited anabnormal clinical assessment for at least one day from D1 to D177. Pig120 exhibited coughing from D5-7 and on D12. Pig 126 was thin fromD2-18, exhibited depression from D4-D5, on D10, and from D17-D19, roughhair coat on D5, and labored respiration from D18-D22. Pig 132 exhibitedan abscess from D49-D56. Pig 145 exhibited a swollen sheath from D37-D43and from D46 to D74, and a sore on the sheath from D75-D83 and from D85to D87. Pig 151 exhibited lameness and/or a swollen leg from D78-D83 andon D85. Pig 155 exhibited an abscess on D69-D77.

Three mortalities occurred prior to challenge. Pig 179 (PRRS 94881 MLV,D6): Necropsy revealed minimal lesions (thin, poor body condition).Laboratory testing showed mild macrophagic interstitial pneumonia.Immunohistochemistry was negative for PRRS. Intestinal samples wereautolyzed but did not show evidence of severe necrosis or severeinflammation. Smooth Escherichia coli and Enterococcus spp were isolated(Section 15.9). Pig 124 (Challenge control, D21): No gross lesions wereidentified at necropsy. Laboratory testing revealed severe suppurativeto pyogranulomatous meningoencephalitis with suppurative perivasculitis.Marked pulmonary and hepatic congestion were also evident. The diagnosiswas Streptococcus suis associated meningoencephalitis. Pig 161(Challenge control, D10): Necropsy revealed minimal lesions (thin, poorbody condition). Bordetella bronchiseptica, Streptococcus alphahemolytic and Staphylococcus auricularis were isolated from lungtissues.

PRRS Serology

All pigs were PRRS ELISA negative on D0 and D7. By D14, 90% of PRRS94881 MLV-vaccinated pigs had positive PRRS ELISA titers. This numberincreased to 95% on D21 and was 100%, 100%, 100%, 90%, 100% and 95% onD28, D56, D84, D112, D140 and D168, respectively. None of the challengecontrol pigs developed PRRS antibody titers during the vaccination phaseof this study, and from D14 through D168, a significantly higherpercentage of PRRS 94881 MLV-vaccinated pigs had positive PRRS antibodytiters compared to challenge controls (p<0.0001).

During the challenge phase of the study, the percentages of PRRS 94881MLV-vaccinated pigs with positive PRRS ELISA titers were 95%, 95%, 100%and 100% on DPC 0, DPC 3, DPC 7 and DPC10, respectively. In contrast,challenge control pigs did not develop PRRS antibody titers until DPC 7,when 30% had titers. This increased to 80% on DPC 10. The PRRS 94881MLV-vaccinated pigs had higher percentages of animals with positive PRRSantibody titers throughout the challenge phase of the study (p≦0.0478).

Pigs in the negative control group were PRRS ELISA seronegativethroughout the study with the exception of two pigs on D112. Numbers 116and 120 were PRRS ELISA seropositive on D112.

A summary of group percentages of pigs with positive PRRS-antibodytiters before challenge is shown below in Table 8.28. Data from thechallenge portion of the study are shown below in Table 8.29.

TABLE 8.27 Summary of Group Frequency of Pigs with PositivePRRS-Antibody Titer by Day, Days 0-168 N % Pos- Total Day Group¹Positive itive 95% CI Number p value 0 1 0 0 0.0 15.4 22 NA 2 0 0 0.015.4 22 3 0 0 0.0 26.5 12 NI 7 1 0 0 0.0 16.1 21 NA 2 0 0 0.0 15.4 22 30 0 0.0 26.5 12 NI 14 1 19 90 69.6 98.8 21 <0.0001 2 0 0 0.0 16.1 21 3 00 0.0 26.5 12 NI 21 1 20 95 76.2 99.9 21 <0.0001 2 0 0 0.0 16.8 20 3 0 00.0 26.5 12 NI 28 1 21 100 83.9 100.0 21 <0.0001 2 0 0 0.0 16.8 20 3 0 00.0 26.5 12 NI 56 1 21 100 83.9 100.0 21 <0.0001 2 0 0 0.0 16.8 20 3 0 00.0 26.5 12 NI 84 1 21 100 83.9 100.0 21 <0.0001 2 0 0 0.0 16.8 20 3 0 00.0 26.5 12 NI 112 1 19 90 69.6 98.8 21 <0.0001 2 0 0 0.0 16.8 20 3 2 172.1 48.4 12 NI 140 1 21 100 83.9 100.0 21 <0.0001 2 0 0 0.0 16.8 20 3 00 0.0 26.5 12 NI 168 1 20 95 76.2 99.9 21 <0.0001 2 0 0 0.0 16.8 20 3 00 0.0 26.5 12 NI ¹Group 1 = PRRS 94881 MLV vaccine; Group 2 = Challengecontrol group; Group 3 = Negative control group. NI = Not included instatistical analysis. NA = Not applicable, no analysis conducted

TABLE 8.29 Summary of Group Frequency of Pigs with PositivePRRS-Antibody Titer by Day, DPC 0 through DPC 10 N % Pos- Total DayGroup¹ Positive itive 95% CI Number p value D 179 1 20 95 76.2 99.9 21<0.0001 (DPC 2 0 0 0.0 16.8 20 0) 3 0 0 0.0 26.5 12 NI D 182 1 20 9576.2 99.9 21 <0.0001 (DPC 2 0 0 0.0 16.8 20 3) 3 0 0 0.0 26.5 12 NI D186 1 21 100 83.9 100.0 21 <0.0001 (DPC 2 6 30 11.9 54.3 20 7) 3 0 0 0.026.5 12 NI D 189 1 21 100 83.9 100.0 21  0.0478 (DPC 2 16 80 56.3 94.320 10) 3 0 0 0.0 26.5 12 NI ¹Group 1 = PRRS 94881 MLV vaccine; Group 2 =Challenge control group; Group 3 = Negative control group. NI = Notincluded in statistical analysis.

Discussion/Conclusion

To achieve the study objective, twenty-two (22) healthy, PRRSsusceptible and seronegative pigs were inoculated IM with 1 mL of PRRS94881 MLV at approximately 14 days of age. Thirty-four (22pigs—challenge control group and 12 pigs—negative control group) PRRSsusceptible and seronegative pigs were inoculated IM with 1 mL ofcontrol product at approximately 14 days of age.

Validation of the Study and Challenge Model

Pigs in the negative control group remained negative for PRRSv (viremia;qPCR) throughout the study. Two pigs (Nos. 116 and 120) in the negativecontrol group had positive ELISA titers on D112, while all other ELISAresults for this group were negative. Considering that no viremia wasdetected in these pigs or in the group as whole; likewise, all otherserum samples were ELISA negative, the results for these two pigs onD112 were considered false positives possibly due to an unassignable laberror. Thus, this was a valid study. Unrelated to the establishment of avalid study, the negative control group had a median histological lunglesion score of 9.0 on D189 in contrast to a median gross lesion scoreof 0.0%. These data highlight that pigs housed under normal swinehusbandry conditions for an extended period of time develop minor lunglesions that are inconsequential and not related to specific pathogens.

Following inoculation with European PRRS isolate 205817 by the methoddescribed earlier, the challenge control group exhibited a mean ADWGfrom DPC 0 to DPC 9 of 0.2 kg/day (a mean ADWG of 0.5 kg/day for thenegative control group), a median gross lung lesion score of 13.8% (0.0%for the negative control group), a median histological lung lesion scoreof 19.5 (9.0 for the negative control group) and a median value of 6.25log₁₀ GE/mL for the detection of PRRSv RNA in lung tissue (median of 0.0log₁₀ GE/mL for negative control group). These results highlight thatPRRS-specific clinical disease was induced in the challenge controlgroup, thus validating this challenge model as an adequate clinicallaboratory tool to evaluate PRRS vaccine efficacy and more specifically,26 week duration of immunity of PRRS 94881 MLV.

Determination of 26 Week Duration of Immunity of PRRS 94881 MLV

Determination of DOI for PRRS 94881 MLV of 26 weeks post-vaccination wasbased upon the vaccine group exhibiting a significant reduction (p≦0.05)in post-challenge lung lesions (gross or histological) compared with thechallenge control group.

Gross and histological lung lesions were selected as the primaryparameter for determination of 26 week DOI because this parameterprovides the most clinically relevant and convincing evidence ofefficacy when evaluating a new vaccine within the PRRS respiratorychallenge model in pigs. Lung lesion development is one of the hallmarksof PRRS respiratory disease in pigs and can be considered the source forall subsequent manifestations of secondary PRRSv disease characteristicssuch as clinical signs, pyrexia, decreased ADWG, etc.

The PRRS 94881 MLV group exhibited a significant reduction in gross lungpathology post-challenge, as evidenced by a median gross lung lesionscore of 0.1% in comparison to the challenge control group, whichexhibited a median gross lung lesion score of 13.8% (p<0.0001). Inaddition, the PRRS 94881 MLV group exhibited a significant reduction inhistological lung lesion scores, as evidenced by a median histology lunglesion score of 6.0 for the PRRS 94881 MLV group compared with a medianhistology lung lesion score of 19.5 for the challenge control group(p<0.0001). Thus, DOI of 26 weeks for PRRS 94881 MLV at dosage of1×10^(4.27) TCID₅₀ was established based upon the primary parameter of asignificant reduction for lung lesions post-challenge. This result wasachieved with a vaccine dose slightly lower than the targeted minimumimmunizing dose of 1×10^(4.5) TCID₅₀/mL. One challenge control pig (No.123) could not be scored for gross lung lesions because of pleuritis andadhesions due to bacterial infections, but was scored for histologicallung lesions. The omission of this pig from the challenge controlgroup's gross lung lesion score analysis did not affect the outcome ofthis study.

Viremia post-challenge was selected as the most important secondaryparameter because it represents the level of viral replication andpersistence occurring within the host animal upon exposure. Asignificant reduction (p≦0.05) in viremia would correspond with a PRRSvaccine that induces adequate immunity to limit PRRS pathogenesis withinthe host. At 3, 7 and 10 days post-challenge, the PRRS 94881MLV-vaccinated group demonstrated a significant reduction in viremia(qPCR) compared with the challenge control group (p<0.0001). To furtherevaluate post-challenge viremia between groups, the quantity of theviral load over a specific duration of time post-challenge wascalculated, as represented as “area under curve” or AUC. The PRRS 94881MLV-vaccinated group had a median AUC value from DPC 0 to DPC 10 of15.54 log₁₀ GE/mL/day; while the challenge control group had a medianAUC value of 44.77 log₁₀ GE/mL/day (p<0.0001). In addition, the PRRS94881 MLV-vaccinated group had a median AUC value from DPC 3 to DPC 10of 8.88 log₁₀ GE/mL/day; while the challenge control group had a medianAUC value for this period of 36.43 log₁₀ GE/mL/day (p<0.0001). Whetherviremia was examined at specific time points post-challenge or over thecourse of the post-challenge period, PRRS 94881 MLV administered 26weeks prior to challenge with a virulent heterologous European strain ofPRRS significantly (p≦0.05) reduced viremia after challenge inoculation.

In association with a reduction of PRRS viremia post-challenge, asignificant (p≦0.05) reduction in the viral load in lung tissue wouldalso be of great importance from the standpoint of PRRS vaccineimmunity. A reduction of viral load in the lung tissue may be associatedwith reduced viral stability, replication and persistence within thehost and may secondarily lead to reduced shedding of PRRSv to otherpigs. In this study, lung tissues from the PRRS 94881 MLV group had amedian lung qPCR result of 3.69 log₁₀ GE/mL 10 days post challenge (DPC10) while the challenge control group had a median lung qPCR result of6.25 log₁₀ GE/mL. The difference between the vaccine group and thechallenge control group was significant (p<0.0001), thus furthersupporting duration of immunity of 26 weeks.

A marked reduction in severity and frequency of clinical signspost-challenge in pigs would also be supportive of PRRS vaccine efficacyand establishment of DOI of 26 weeks for PRRS 94881 MLV. Only one pigexhibited clinical signs following challenge: pig 149 (challengecontrol) had a respiratory score of “1” (panting/rapid respiration) onD185. No pigs in the PRRS 94881 MLV-vaccinated group exhibited clinicalsigns during the post-challenge phase of this study and there were nostatistical differences between the vaccinated and challenge controlgroups (p=0.4878 or no test conducted). Clinical signs post-challengewere not strong enough in this study to assess the DOI.

Pyrexia between groups varied post-challenge. PRRS 94881 MLV-vaccinatedpigs exhibited significantly lower LS Mean rectal temperatures on twodays (DPC 8 and DPC 10; (p≦0.0183) and higher LS Mean rectaltemperatures on four days (DPC 0, DPC 2, DPC 4 and DPC 5; p≦0.0281)compared with challenge control pigs. Otherwise, no significantdifferences were detected between groups post-challenge (p≧0.0642).Although statistical differences between groups were detectedpost-challenge, these differences were not biologically meaningful,considering that mean rectal temperatures remained ≦39.9° C. (challengecontrol group, D182) for all groups. No difference was detected betweengroups with respect to the proportion of pigs with pyrexia for at leastone day post-challenge (p=0.4537).

The presence of significant viremia, lung pathology and viral load inlung tissues due to PRRS in the challenge control group resulted in asignificant (p≦0.05) difference between groups for body weight on DPC 9.In this study, the vaccinated and challenge control groups had LS meanbody weights on DPC 9 of 138.3 kg and 130.3 kg, respectively (p=0.0455).The LS mean ADWG from DPC 0 to DPC 9 were 0.4 kg/day and 0.2 kg/day, forvaccinated and challenge groups, respectively. This difference was notstatistically significant (p=0.1041).

Post-Vaccination Parameters Examined in this Study

Three pigs were found dead during the vaccination phase of this study.Pig 179 (PRRS 94881 MLV-vaccinated) was found dead on D6 associated withsmooth Escherichia coli and Enterococcus spp. infections. Pig 161(challenge control group) was found dead on D10 associated withBordetella bronchiseptica, Streptococcus alpha hemolytic andStaphylococcus auricularis infections. Pig 124 (challenge control group)was found dead on D21 associated with a Streptococcus suis infectionthat lead to meningoencephalitis. To control and prevent any moredeaths, pigs were mass treated with injectable vitamins and antibiotics.Following treatments, no more deaths occurred. Since deaths occurred inboth treatment groups it can be assumed that the IVP itself was notassociated with infections. More likely, pigs arrived at the researchfacility harboring these infections. Data for these pigs were includedwhen available. Gross and histological lung lesion scores from thesepigs were omitted from lung lesion analyses since these pigs died beforechallenge administration. The loss of one—PRRS 94881 MLV pig andtwo—challenge control pig during the extended time period fromvaccination to challenge did not affect the outcome of the study.

No abnormal clinical assessments related to PRRS 94881 MLV vaccinationor control product were observed in pigs following inoculation on D0.Seven pigs in the PRRS 94881 MLV-vaccinated group had abnormalassessments post-vaccination; while thirteen challenge control pigs hadabnormal assessments. Excluding the three pigs that died due tobacterial infections, these abnormal assessments included thinness,coughing, swellings, rough hair coat, depression, abscesses and poorbody condition at various time points, none of which lasted an extendedperiod of time. In the author's opinion, these findings were not relatedto the administration of either experimental product, but rather aretypical findings in growing/maturing pigs, under group housingsituations, over an extended period of time.

All pigs were PRRS ELISA serology negative on D0, thus confirming thatall pigs met the inclusion criterion of being PRRS sero-negative uponentry into the study. The majority of pigs (90%) receiving PRRS 94881MLV sero-converted to PRRS by D14 and all PRRS-vaccinated pigs wereseropositive by D28. Conversely, the challenge control pigs remainedseronegative until 7 days post-challenge, when this group began todemonstrate PRRS seroconversion. As covered earlier, two—negativecontrol pigs were PRRS ELISA seropositive on D112, which was consideredan incidental finding, possible due to an unassignable lab error.

At 7, 14, 21 and 28 days post-vaccination, the PRRS 94881 MLV-vaccinatedgroup exhibited median qPCR results of 3.00, 0, 0 and 3.00 log₁₀ GE/mL,respectively. These results highlight that within 4 weekspost-vaccination, a dosage of 1×10^(4.27) TCID₅₀ of PRRS 94881 MLVinduced sufficient replication of the MLV that is often required tobuild protective immunity already at 4 weeks after vaccination.Conversely, the challenge control group was negative for PRRS viremiauntil three days post-challenge.

CONCLUSION

A significant reduction (p≦0.05) of gross and histological lung lesionsat necropsy, viral load in lung tissues at necropsy and viremiapost-challenge for the PRRS 94881 MLV group compared to challengecontrol group demonstrated vaccine efficacy against virulent PRRSv whenvaccinated at 2 weeks of age and challenged 26 weeks post-vaccination.The results of this study therefore support the demonstration ofduration of immunity of 26 weeks post-vaccination with PRRS 94881 MLV.These results were achieved with a vaccine dose of 1×10^(4.27)TCID₅₀/mL, which was slightly lower than the minimum immunizing dose(1×10^(4.5) TCID₅₀/mL).

The sequences of the PRRSV 94881 attenuated strain and the parentalstrain are as follow:

SEQ ID NO: 1: FULL LENGTH NUCLEOTIDE SEQUENCE OF PRRS Master SeedVirus of 94881 1TTTGTGTACC TTGGAGGCGT GGGTACAGCC CTGCCCCACC CTTTGGTCCC TGTTCTAGCC 61CGACAAGTAC CCTTCTCTCT CGGGGCGAGC GCGCCGCCTG CTGCTCCCTT GCGGCGGGAA 121GGACCTCCCG AGTATTTCCG GAGAGCACCT GCTTTACGGG ATCTCCGCCC TTTAACCATG 181TCTGGGATGT TCTCCCGGTG CATGTGCACC CCGGCTGCCC GGGTATTTTG GAACGCCGGC 241CAAGTCTATT GCACACGGTG TCTCAGTGCA CGGTCTCTTC TCTCTCCAGA ACTTCAGGAC 301ACGGACCTCG GTGCAGTTGG CTTGTTTCAC AAGCCTAAAG ACAAGCTCCA TTGGAAAGTT 361CCCATTGGTA TCCCCCAGGT GGAATGTTCT CCATCTGGGT GTTGCTGGCT GTCAACCATT 421TTTCCTTTAG CGCGCATGAC CTCCGGCAAT CACAACTTCC TTCAACGACT CGTGAAGGTT 481GCTGATGTAT TGTACCGTGA CGGTTGCTTA ACCCCTAGAC ACCTCCGTGA ACTCCAAGTT 541TACGAGCGTG GTTGCAATTG GTATCCGATT ACGGGGCCTG TGCCTGGGAT GGCTGTGTAC 601GCGAACTCCA TGCACGTGTC CGACCAACCG TTCCCTGGTG CCACTCATGT GTTAACAAAT 661TCCCCTTTGC CTCAACGGGC TTGTCGGCAG CCGTTCTGTC CGTTCGAAGA GGCCCATTCT 721AGCATATACA GGTGGGAAAA ATTTGTAATT TTTATGGATT CCTCCTCCGA CGGTCGATCT 781CGCATGATGT GGACTCCGGA ATCCGATGAC TCCACGGCTT TGGAAGTTCT GCCGCCCGAG 841CTAGAACACC AGGTCAAGGT CCTTGTTCGG AGCTTTCCCG CCCATCACCT TGTCGACCTT 901GCCGATTGGG AGCTCACTGA GTCCCCTGAT AACGGTTTTT CCTTCAGCAC GTCACATCCT 961TGCGGCTACC TTGTTCGGGA CCCGGCTGTA TCCGAAGGCA AGTGTTGGCT TTCCTGCTTT 1021TTGAGCCAGT CAGCCGAAGT GCTCAGTCGC GAGGCGCATC TGGCTACCGC CTATGGTTAC 1081CAAACCAAGT GGGGTGTGCC TGGCAAGTAC ATCCAGCGCA GACTTCAAGT TCACGGTCTC 1141CGTGCTGTGG TCGACCCTGA TGGTCCCATT CACGTTGAAG CATTGTCTTG CCCCCAGTCT 1201TGGATCAGGC ACTTGACCCT GAATGATGAT GTCACCCCGG GATTCGTTCG CCTAATGTCT 1261CTTCGCATTG TGCCGAACAC AGAGCCTACC ACACACCGGA TCTTTCGTTT TGGAGTGCAC 1321AAGTGGTATG GTGCCGCCGG CAAACGGGCC CGTGGCAAGC GTGCCGCCAA AAGTGAGAAA 1381GACTCGGCTT CCACCCTCAA GGTTGCCCGA CCGACTTCCA CCAGTGGAAT CGTCACCTAC 1441TCCCCACCTG CGGACGGGTC TTGTGGTTGG CATGCCCTTG CCGCCATACT GAACCGGATG 1501ATTAATAATG ACTTCACGTC CCCTCTGCCT CGGTACAACA GGCCGGAGGA CGATTGGGCT 1561TCTGATGGTG ACCTTGCTCA GGCCATTCAA TGTTTGCAAC TACCTGCCGC CATAGCTCGG 1621AACCGCGCCT GCCCTAACGC CAAATACCTC ATAAAACTCA ACGGAGTTCA TTGGGAGGTA 1681GAGGTGAGGC CTGGAATGGC TCCTCGCTCC CTCTCTCGTG AGTGCGTTGT TGGCGTCTGC 1741TCTGAAGGCT GTGTCGCGTC GCCTTACCCG GAGGACGGGT TGCCTAAACG TGCACTTGAG 1801GCCCTGGCGT CTGCTTATAG ACTGCCTTCA GACTGTGTTT GTGATGGTAT TATTGACTTC 1861CTTGCCAATC CACCTCCCCA GGAGTTCTGG ACTCTTGACA AAATGTTGAC TTCCCCGTCA 1921CCGGAGCAGT CCGGCTTCTC TAGTCTGTAT AAATTGTTGT TAGAGATCTT GCCGCAGAAA 1981TGCGGATCCA CAGAAGGGGA ATTCATCTAT ACTGTTGAGA GGATGTTGAA GGATTGTCCG 2041AGCTCCAAAC AGGCCATGGC CCTCCTTGCA AAAATTAAGG TCCCATCCTC AAAGGCCCCA 2101TCCGTGACTC TGAACGAGTG CTTCCCCACG GATGTTCCAG TCAACTCTGA GTTAATATCT 2161TGGGAAGAGC CCAAAGACCC TGGCGCTGCT GTTGTCCTAT GTCCATCGGA TGCAAAAGAA 2221TCTAAGGAAA CAGCCCCTGA AGAAGCTCAA GCGAGAAACC GTAAGGTCCT TCACCCTGTG 2281GTCCTTACCG AGGAACTTAG CGAGCAACAG GTGCAGGTGG TTGAGGGTGA TCAGGATATG 2341CCACTGGATT TGACTTGGCC AACCTTAACC GCTACGGCGA CCCCTGTTAG AGGGCCGGTA 2401CCGGACAATT TGAGCTCTGG CATTGGTGCC CAGCCCGCTA CCGTTCAAGA ACTCATTCTG 2461GCGAGGCCTG CACCCCGTCT TGTTGAGCGC TGTGGCACGG AGTCGAACGG CAGCAGTTCA 2521TTTCTGGATT TGCCTGACGT GCAGACCTCG GACCAGCCTT TAGACCTGTC CCTGGCCGCG 2581TGGCCTGTAA GGGCTACCGC GTCTGACCCC GGTTGGATCC ACGGTAGGCG TGAGCCTGTC 2641TTTGTGAAGC CTCGAGGTGT TTTCTCTGAT GGCGAGTCGG CCCTTCAGTT CGGAGAGCTT 2701TCCGAAGCCA GTTCTGTCGT CGATGACCGG ACAAAAGAAG CTCCGGTGGT TGACGCCCCC 2761ATCGATTTGA CAACTTCGAA CGAGACGCTC TCTGGGTCTG ACCCCTTTGA ATTCGCCAAA 2821TTCAGGCGCC CGCGTTTCTC CGCGCAAGCT TTAATCGACC GAGGTGGTCC GCTTGCCGAT 2881GTTCATGCAA AGATAAAGAG TCGGGTATAT GAACAATGCC TTCAAGCTTG TGAACCTGGT 2941AGTCGTGCGA CCCCAGCCAC CAAGAAGTGG CTCGACAAAA TGTGGGACAG GGTGGACATG 3001AAAACTTGGC GCTGCACCTC GCAGTTCCAA GCTGGTCACA TTCTTGAGTC CCTCAAATTC 3061CTCCCTGACA TGATTCAAGA CACACCGCCT CCTGTTCCCA GGAAGAACCG AGCTGGTGAC 3121AGTGCCGGCC TGAAGCAACT GGTGGCGCAG TGGGATAGGA AATCGAGTGT GACACCCCCC 3181ACAAAACCGG TTGGACCGGT GCTTGACCAG GCCGTCCCTC TGCCTATGGA CATCCAGCAA 3241GGAGATGCCA TCTCCGCTGA CAAGCCACCC CATTCGCAAA ACCCTTCTAG TCAAGTAGAT 3301GTGGGTGGAG GTTGGAAAAG TTTTATGCTC TCCGGCACCC GTTTCGCGGG GTCCGTTAGT 3361CAGCGCCTTA CGACATGGGT TTTTGAGGTT CTCTCCCATC TCCCAGCTTT TATGCTCACA 3421CTTTTCTCGC CACGGGGCTC TATGGCTCCA GGTGATTGGC TGTTTGCAGG TGCTGTTCTA 3481CTTGCTCTCC TGCTCTGCCG TTCTTACCCA ATACTCGGAT GCCTTCCCTT ATTGGGTGTC 3541TTTTCTGGTT CTGTGCGGTG TGTTCGTTTG GGTGTTTTTG GTTCTTGGAT GGCTTTTGCT 3601GTATTTTTAT TCTCGACTCC ACCCGACCCA GTCGGTTCTT CTTGTGACCA CGATTCGCCG 3661GAGTGTCATG CTGAGCTTTT GGCTCTTGAG CAGCGCCAAC TTTGGGAACC TGTGCGCAGC 3721CTTGTGGTCG GGCCATCGGG CCTCTTATGC GTCATTCTTG GCAAGTTACT CGGTGGGTCA 3781CGTTGTCTCT GGTTTGTTCT CCTACGTATA TGCATGCTCG CAGATTTGGC AATTTCTCTT 3841ATTTATGTGG TGTCCCAAGG GCGTTGTCAC AAGTGTTGGG GAAAGTGTAT AAGGACGGCT 3901CCTGCAGAAG TGGCCCTTAA TGTGTTTCCT TTTTCGCGCG CCACCCGCTC ATCTCTTGTG 3961TCCTTGTGTG ATCGGTTCCA AGCGCCAAAA GGAGTTGACC CCGTGCACTT GGCGACAGGC 4021TGGCGCGGGT GCTGGTGTGG TGAGAGCCCT ATTCATCAAT CACACCAAAA ACCGATAGCT 4081TATGCCAACT TGGATGAAAA GAAGATATCC GCCCAGACGG TGATTGCTGT CCCGTATGAT 4141CCTAGTCAGG CCATTAAATG CCTGAAAGTT TTGCAGGCAG GAGGGGCTAT TGTGGACCAG 4201CCTACGCCCG AGGTCGTCCG TGTGTCTGAG ATTCCCTTCT CGGCCCCATT TTTTCCGAAG 4261GTCCCAGTCA ACCCAGACTG CAGGGTTGTG GTAGATTCGG ACACTTTTGT GGCTGCGGTC 4321CGCTGCGGTT ATTCGACAGC ACAACTGGTC CTTGGTCGGG GCAACTTTGC CAAGCTAAAT 4381CAGACCCCCC TCAGGAACTC TGTCCCCACC AAAACAACTG GTGGGGCCTC ATACACCCTT 4441GCCGTGGCCC AGGTATCTGT GTGGACTCTT GTTCATTTCA TCCTCGGCCT TTGGTTAACG 4501TCACCTCAAG TGTGTGGTCG AGGGACCTCT GACCCGTGGT GTTCGAACCC TTTTTCGTAT 4561CCTACTTATG GCCCCGGAGT TGTGTGTTCC TCTCGACTCT GCGTGTCTGC CGACGGAGTT 4621ACCCTGCCAT TGTTCTCAGC CGTTGCCCAT CTTTCCGGTA GAGAGGTGGG GATTTTTATT 4681TTGGTGCTTG CCTCCTTGGG CGCTTTAGCC CACCGCTTGG CTCTTAAGGC AGACATGTCA 4741ATGGTCTTTT TGGCGTTTTG TGCTTACGCC TGGCCCATGA GCTCCTGGTT AATTTGCTTC 4801TTTCCTATGC TCTTGAGGTG GGTAACCCTT CATCCTCTCA CTATGCTTTG GGTGCACTCA 4861TTTTTGGTGT TTTGCCTACC AGCTGCCGGC GTTCTCTCGC TGGGAATAAC CGGTCTTCTT 4921TGGGCAGTTG GCCGTTTCAC CCAGGTTGCC GGAATTATCA CACCTTATGA CATCCACCAG 4981TATACCTCCG GACCACGTGG TGCAGCTGCT GTAGCAACGG CTCCAGAAGG TACTTACATG 5041GCGGCCGTTC GGAGAGCCGC TTTGACTGGA CGGACTTTGA TCTTCACACC ATCTGCAGTC 5101GGATCCCTTC TTGAAGGTGC TTTCAGAACT CAAAAGCCCT GCCTTAACAC CGTGAATGTC 5161GTAGGCTCTT CCCTTGGTTC TGGAGGAGTT TTCACCATTG ATGGCAGAAG AGTCATCGTC 5221ACTGCCACCC ATGTGTTGAA TGGTAACACA GCCAGGGTCA CTGGTGATTC CTACAACCGC 5281ATGCACACGT TCAATACTAA TGGTGATTAT GCCTGGTCCC ATGCTGATGA CTGGCAAGGC 5341GTTGCCCCTA TGGTTAAGAT CGCTAAGGGG TATCGCGGTC GTGCCTACTG GCAAACGTCA 5401ACCGGAGTCG AACCTGGCAT CATGGGGGAA GGATTCGCCT TCTGTTTCAC TAACTGTGGC 5461GACTCAGGGT CACCTGTCAT TTCAGAAGCT GGTGACCTTA TTGGAGTCCA TACCGGTTCA 5521AACAAACTCG GTTCTGGTCT TGTGACAACC CCTGAAGGGG AGACCTGCTC CATCAAGGAA 5581ACTAGGCTCT CTGACCTTTC TAGACATTTT GCAGGTCCAA GCGTCCCTCT TGGGGACATT 5641AAGTTGAGCC CAGCCATCAT CCCTGATGTG ACAACTATTC CGAGTGACTT GGCATCGCTC 5701CTTGCTTCTG TCCCCGTGAT GGAAGGTGGC CTCTCAACTG TCCAGCTTTT GTGCGTCTTT 5761TTCCTTCTCT GGCGCATGAT GGGCCATGCC TGGACACCCA TTGTTGCCGT AGGCTTCTTT 5821TTGCTGAATG AAATTCTCCC AGCAGTCTTG GTCCGAGCTG TGTTCTCTTT TGCACTCTTT 5881GTACTTGCAT GGGCCACCCC CTGGTCGGCA CAAGTGTTGA TGATTAGACT CCTCACGGCG 5941GCTCTCAACC GCAACAGGTT GTCCCTGGCG TTCTACGCAT TCGGAGGTGT CGTTGGCCTG 6001GCCACAGAAA TCGGGACTTT TGCTGGTGGA TGGCCTGAAC TGTCCCAAGC CCTCTCGACA 6061TACTGCTTCC TGCCCAGGTT CCTTGCTGTG ACTAGTTATG TCCCCACCAT CATCATCGGT 6121GGGCTCCATG CCCTCGGCGT AATTTTGTGG TTATTCAAAT ACCGATGCCT CCACAACATG 6181CTGGTTGGTG ATGGGAGTTT CTCAAGCGCT TTCTTCCTAC GGTATTTTGC TGAGGGTAAT 6241CTTAGGAAAG GCGTGTCGCA GTCCTGTGGC ATGAATAACG AATCCCTGAC AGCTGCTTTG 6301GCTTGCAAGT TGTCGCAAGC TGACCTTGAT TTTTTGTCCA GTTTAACGAA CTTCAAGTGC 6361TTTGTGTCCG CTTCAAACAT GAAAAATGCA GCTGGCCAAT ACATCGAGGC GGCGTATGCT 6421AGAGCTCTGC GTCAGGAGCT GGCCTCCTTG GTTCAGGTTG ACAAGATGAA AGGAGTATTG 6481GCCAAGCTCG AGGCTTTCGC TGAGACGGCC ACTCCGTCAC TTGACACAGG GGACGTGATT 6541GTTCTGCTTG GGCAACACCC CCATGGATCC ATCCTCGACA TTAATGTGGG GGGTGAAAGG 6601AAAACTGTGT CTGTGCAAGA AACACGATGC CTGGGTGGTT CCAAATTCAG TGTCTGCACT 6661GTCGTGTCCA ACACGCCCGT GGATACCTTG ACCGGTATCC CACTTCAGAC GCCAACCCCA 6721CTTTTTGAAA ATGGCCCGCG CCATCGCAGC GAGGACGACG ACCTCAAAGT TGAGAGAATG 6781AAAAAACACT GTGTATCCCT CGGCTTCCAC AAAATCAATG GTAAAGTTTA CTGCAAAATT 6841TGGGACAAGT CTAACGGCGA CACCTTTTAC ACGGATGATT CCCGATACAC TCAAGACCAT 6901GCTTTTCAGG ACAGGTCAAC CGACTATAGA GACAGGGATT ATGAAGGTGT ACAGACCGCC 6961CCCCAACAGG GATTCGATCC AAAGTCCGAA GCCCCTGTTG GCACTGTTGT AATCGGTGGC 7021ATTACGTATA ACAGGCATCT GGTCAAAGGT AAGGAGGTCC TAGTTCCCAA ACCTGACAAC 7081TGCCTTGAAG CTGCCAGACT GTCCCTTGAG CAAGCTCTTG CTGGGATGGG CCAAACTTGT 7141GACCTTACAG CTACCGAAGT GGAGAAACTA AAGCGCATCA TTAGTCAACT CCAAGGTCTG 7201ACCACTGAAC AGGCTTTAAA CTGCTAGCCG CCAGCGGCTT GACCCGCTGT GGCCGCGGCG 7261GCCTAGTTGT AACTGAAACG GCGGTAAAAA TCGTAAAATA CCACAGCAGA ACTTTCACCT 7321TAGGCTCTTT AGACCTAAAA GTCACCTCCG AGGTGGAGGT GAAGAAATCA ACTGAGCAGG 7381GGCACGCTGT CGTGGCGAAC TTATGTTCCG GTGTCGTCTT GATGAGGCCT CACCCACCGT 7441CCCTTGTTGA CGTTCTCCTC AAACCCGGAC TTGACACAAC ACCCGGCATT CAACCAGGGC 7501ATGGGGCCGG GAATATGGGC GTGAACGGTT CTATTTGGGA TTTTGAAACT GCACCCACAA 7561AGGTAGAACT AGAGTTGTCC AAGCAAATAA TCCAAGCATG TGAAGTCAGG CGCGGGGACG 7621CCCCTAACCT CCAACTCCCC TACAAGCTTT ATCCTGTCAG GGGGGACCCC GAGCGGCGTA 7681AAGGTCGCCT TGTCAACACT AGGTTTGGAG ATTTACCTTA CAAAACTCCC CAAGACACCA 7741AGTCCGCAAT TCATGCGGCT TGTTGCCTGC ATCCCAATGG GGTCCTCGTG TCTGATGGCA 7801AATCCACGCT GGGTACCACT CTTCAACATG GTTTCGAGCT TTATGTCCCC ACTGTACCTT 7861ATAGTGTCAT GGAATACCTT GATTCACGCC CTGACACCCC TTTTATGTGT ACTAAACATG 7921GCACTTCCAA GGCTGCTGCA GAGGACCTCC AAAAATATGA CCTATCCACT CAAGGGTTTG 7981TCTTGCCTGG GGTCCTACGC CTAGTGCGCA GGTTCATCTT TAGCCATGTT GGTAAGGCGC 8041CACCACTGTT CCTTCCATCA ACCTACCCTG CCAAGAACTC CATGGCAGGG GTCAATGGCC 8101AGAGGTTCCC AACAAAGGAT GTCCAGAGCA TACCTGAAAT TGATGAAATG TGCGCCCGTG 8161CCGTCAAGGA AAATTGGCAG ACTGTGACAC CTTGCACCCT CAAAAAACAG TACTGTTCCA 8221AACCTAAAAC TAGAACCATC CTAGGTACCA ACAACTTCAT AGCCTTGGCT CACAGGTCAG 8281CACTCAGTGG TGTCACCCAG GCGTTCATGA AGAAGGCCTG GAAGTCCCCA ATTGCCTTGG 8341GGAAAAACAA GTTTAAGGAA TTGCATTGCA CTGTCGCCGG CAGATGCCTT GAGGCTGACC 8401TGGCTTCCTG CGATCGCAGC ACCCCCGCCA TTGTGAGGTG GTTTGTTGCC AACCTCCTGT 8461ATGAACTTGC AGGATGTGAA GAGTACTTGC CTAGCTACGT GCTCAACTGT TGCCATGACC 8521TTGTGGCAAC GCAGGATGGC GCTTTCACAA AACGCGGTGG CCTGTCGTCC GGGGACCCCG 8581TCACCAGTGT GTCCAACACC GTCTACTCAC TGATAATTTA CGCCCAGCAC ATGGTGCTTT 8641CGGCCTTGAA GATGGGTCAT GAAATTGGTC TCAAGTTCCT TGAGGAACAG CTCAAATTTG 8701AGGACCTTCT TGAAATCCAG CCCATGTTAG TGTATTCTGA TGACCTCGTC TTGTATGCGG 8761AAAGACCCAC TTTTCCCAAC TACCATTGGT GGGTCGAGCA TCTTGACCTG ATGTTGGGCT 8821TTAAAACGGA CCCAAAGAAA ACTGTCATAA CTGATAAACC CAGTTTTCTC GGCTGCAGAA 8881TTGAAGCAGG ACGGCAGTTA GTCCCCAATC GCGACCGTAT TCTGGCTGCT CTTGCATATC 8941ATATGAAGGC GCAGAACGCC TCAGAGTATT ATGCGTCCGC TGCCGCAATT CTGATGGATT 9001CGTGTGCTTG CATTGACCAT GACCCCGAGT GGTATGAGGA TCTTATCTGC GGCATCGCCC 9061GGTGTGCTCG CCAGGACGGT TACCGTTTTC CAGGCCCGGC ATTTTTCATG TCCATGTGGG 9121AGAAGCTGAA AAGTCATAAT GAAGGGAAGA AATGCCGTCA CTGCGGCATC TGCGACGCCA 9181AAGCCGACTA TGCGTCCGCC TGTGGACTTG ATTTGTGTTT GTTCCATTCA CACTTTCATC 9241AACACTGCCC AGTCACTCTG AGCTGTGGCC ACCATGCCGG TTCAAAGGAA TGTTCGCAGT 9301GTCAGTCACC TGTCGGGGCT GGCAAATCCC CCCTTGACGC TGTGCTGAAA CAAATCCCGT 9361ACAAACCTCC TCGTACCATT ATCATGAAGG TGGACAACAA AACAACGACC CTTGACCCGG 9421GAAGATATCA GTCCCGTCGA GGTCTTGTTG CAGTCAAAAG AGGTATTGCA GGTAATGAGG 9481TTGATCTTTC TGATGGAGAC TACCAAGTGG TGCCTCTTTT GCCGACTTGC AAAGACATAA 9541ACATGGTGAA GGTGGCTTGC AACGTACTAC TCAGCAAGTT TATAGTAGGG CCGCCAGGTT 9601CCGGAAAAAC CACCTGGCTA CTGAACCAAG TCCAGGACGA TGATGTCATT TACACACCTA 9661CTCATCAGAC AATGTTTGAC ATAGTCAGTG CTCTTAAAGT TTGCAGGTAT TCCATCCCAG 9721GAGCCTCAGG ACTCCCTTTT CCACCACCTG CCAGGTCCGG GCCGTGGGTT AGGCTCATCG 9781CCAGCGGACA TGTCCCTGGC CGAGTGTCAT ATCTCGATGA GGCAGGATAT TGCAATCATC 9841TAGACATTCT AAGGCTGCTT TCCAAAACAC CCCTTGTGTG TTTGGGTGAC CTTCAGCAAC 9901TTCACCCGGT CGGCTTTGAT TCCTATTGTT ATGTGTTCGA TCAGATGCCT CAGAAGCAGC 9961TGACCACCAT TTATAGATTT GGCCCTAACA TCTGTGCAGC CATCCAGCCT TGTTACAGGG 10021AGAAACTTGA ATCCAAGGCC AGGAACACCA GAGTGGTTTT CACCACCCGG CCTGTGGCCT 10081TTGGTCAGGT CCTGACACCG TACCACAAAG ATCGTACCGG CTCTGCAATA ACTATAGATT 10141CATCCCAGGG GGCGACCTTC GACATTGTGA CATTGCATCT ACCATCGCCA AAGTCCCTAA 10201ACAAATCCCG AGCACTTGTA GCCATCACTC GGGCAAGACA TGGGTTGTTC ATTTATGACC 10261CTCATGACCA ACTCCAGGAG TTTTTCAACT TAACCCCCGA GCGCACTGAT TGTAACCTTG 10321CGTTCAGCCG TGGGGATGAG CTGGTTGTTT TGAATGTGGA TAATGCGGTC ACAACTGTAG 10381CGAAGGCCCT AGAGACAGGT TCACCCCGAT TTCGAGTATC GGACCCGAGG TGCAAGTCTC 10441TCTTAGCCGC TTGTTCGGCC AGTCTAGAAG GGAGCTGCAT GCCACTACCA CAAGTAGCAC 10501ATAACCTGGG GTTTTACTTT TCCCCGGACA GCCCAGCTTT TGCACCCCTG CCAAAAGAGC 10561TGGCGCCACA TTGGCCAGTG GTCACCCACC AGAATAATCG AGCGTGGCCT GATCGACTTG 10621TCGCTAGTAT GCGCCCAATT GATGCCCGCT ACAGCAAGCC AATGGTCGGT GCAGGGTATG 10681TGGTCGGGCC ATCCATTTTT CTTGGCACTC CTGGTGTGGT GTCATACTAT CTCACATTAT 10741ACATCGGGGG CGAGCCTCAG GCCCTGCCAG AAACACTCGT TTCAACAGGA CGTATAGCCA 10801CAGATTGTCG GGAATATCTC GACGCGGCTG AGGAAGAGGC AGCGAGAGAA CTTCCCCACG 10861CATTTATTGG CGATGTCAAA GGCACTACGA TCGGGGGGTG TCACCACATT ACATCGAAAT 10921ACCTACCTAG GTCCCTGCCT AAAGACTCTG TTGCTGTGGT TGGGGTGAGT TCGCCCGGTA 10981GGGCTGCTAA AGCCGTGTGC ACTCTCACCG ATGTGTACCT CCCCGAACTC CGACCATATT 11041TGCAACCGGA GACGGCATCA AAATGCTGGA AACTTAAACT GGATTTCAGG GATGTTCGAC 11101TGATGGTCTG GAAAGGCGCC ACAGCCTATT TCCAGTTGGA AGGGCTGACA TGGTCAGCGC 11161TGCCCGATTA TGCTAGGTTC ATTCAGCTAC CCAAGGATGC CGTTGTGTAC ATCGATCCGT 11221GTATAGGGCC GGCAACAGCC AATCGCAAGG TTGTGCGAAC CACAGACTGG CGGGCCGACC 11281TGGCAGTGAC ACCGTATGAT TACGGTGCTC AGGTCATTTT GACAACAGCC TGGTTCGAGG 11341ACCTTGGGCC GCAGTGGAAG ATTTTGGGGT TGCAGCCTTT CAGACGAACA TTTGGCTTTG 11401AGAACACTGA AGATTGGGCA ATTCTCGCAC GCCGTATGAA TGACGGCAAA GATTACACTG 11461ACTATAATTG GCATTGTGTA CGAGAACGCC CACACGCAAT TTACGGGCGC GCCCGTGACC 11521ATACGTATCA TTTTGCCCTT GGCACTGAAC TGCAAGTAGA GCTGGGCAGA CCCCGGCTGC 11581CTCCTGAGCA AGTGCCGTGA ACGCGGAGTG ATGCAATGGG TTTACTGTGG AGTAAAATCA 11641GTCAGTTGTT CGTGGATGCC TTCACTGAGT TCCTTGTTAG TGTGGTTGAC ATTGTCATCT 11701TTCTCGCCAT ATTGTTTGGG TTCACTGTTG CAGGCTGGTT ATTGGTCTTC CTTCTCAGAG 11761TGGTTTGCTC CGCGTTTCTC CGTTCGCGCT CTGCCATTCA CTCTTCCGAA CTATCGAAGG 11821TCCTATGAGG GCTTGCTACC CAACTGCAGA CCGGATGTCC CACAATTCGC AGTTAAGCAC 11881CCGTTGGGTA TACTTTGGCA TATGCGAGTC TCCCACCTAA TTGACGAAAT GGTCTCTCGC 11941CGCATTTACC GGACCATGGA ACATTCGGGT CAAGCGGCCT GGAAGCAGGT TGTTAGTGAA 12001GCCACTCTCA CAAAACTGTC AAGGCTTGAC GTAGTCACTC ATTTCCAACA CCTGGCCGCA 12061GTGGAGGCTG ATTCTTGCCG CTTCCTTAGC TCACGACTCG CGATGCTGAA AAACCTTGCC 12121GTTGGCAATG TGAGCCTGGA GTACAACACT ACTTTGGACC GCGTTGAGCT CATCTTTCCC 12181ACACCAGGTA CGAGGCCCAA GTTGACCGAT TTTAGGCAAT GGCTTATCAG CGTGCACGCT 12241TCCATCTTCT CCTCTGTGGC TTCGTCTGTT ACCTTGTTCA CAGTGCTTTG GCTTCGAATT 12301CCAGCTCTAC GCTATGTTTT TGGTTTCCAT TGGCCCACGG CAACACATCA TTCGAACTAA 12361CTATCAATTA CACTATATGT AAGCCATGCC CTACCAGTCA AGCTGCCCAA CAAAGACTCG 12421AGCCTGGCCG TAACGTGTGG TGCAAAATAG GGCACGACAG GTGTGAGGAA CGTGACCATG 12481ATGAGTTGTC AATGTCCATT CCGTCCGGGT ACGACAACCT CAAACTTGAG GGTTATTATG 12541CTTGGCTGGC TTTTTTGTCC TTTTCCTACG CGGCCCAATT CCATCCGGAG CTGTTCGGAA 12601TAGGAAACGT GTCGCGCGTC TTTGTGGATA AGCGACACCA GTTCATTTGC GCCGAGCATG 12661ATGGACAAAA TTCAACCATA TCTGCCAGAC ACAACATCTC CGCGTCGTAT GCGGTGTATT 12721ACCATCATCA AATAGACGGG GGCAATTGGT TTCATTTGGA ATGGCTGCGA CCATTCTTTT 12781CCTCCTGGCT GGTGCTCAAC ATCTCATGGT TTCTGAGGCG TTCGCCTGCA AGCCCTGCTT 12841CTCGACGCAT CTATCAGATA TTAAGACCAA CACGACCGCG GCTGCCGGTT TCATGGTCCT 12901TCAGAACATC AATTGTTTCC AATCTCACAG GGCCTCAACA GCGCAAGGTA CCACTCCCCT 12961CAGGAGGTCG TCCCAATGTC GTGAAGCCGT CGGCATTCCC CAGTACATCA CGATAACGGC 13021TAATGTGACC GATGAATCGT ATTTGTACAA CGCGGACTTG CTGATGCTTT CCGCGTGCCT 13081TTTCTACGCC TCGGAAATGA GCGAGAAAGG CTTCAAAGTC ATCTTTGGGA ATATTTCTGG 13141CGTTGTTTCC GCTTGTGTTA ATTTCACAGA TTATGTGGCC CATGTGACCC AACACACTCA 13201GCAGCACCAT TTGGTAATTG ATCACATTCG GTTACTACAC TTCTTGACAC CGTCTACGAT 13261GAGGTGGGCT ACAACCATTG CTTGTTTGCT TGCCATTCTT TTGGCGGTAT GAAATGTTCT 13321TGCAAGTTGG GGCATTTCTT GACTCCTCAC TCTTGCTTCT GGTGGCTTTT TTTGCTGTGT 13381ACCGGCTTGT CTTGGTCCTT TGTCGATGGC AACGACGACA GCTCGACATC CCAATACATA 13441TATAATTTGA CGATATGCGA GCTGAATGGG ACCGAATGGT TGTCCGGTCA TTTTGATTGG 13501GCAGTCGAAA CCTTTGTGCT TTACCCAGTT GCCACTCATA TCATTTCACT GGGTTTTCTC 13561ACAACAAGCC ATTTCCTTGA TGCGCTCGGT CTCGGCGCTG TGTCCGCCAC AGGATTCATT 13621GGCGAGCGGT ATGTACTTAG CAGCATGTAC GGCGTTTGCG CCTTCGCGGC GTTCGTATGT 13681TTTGTCATCC GTGCTGCTAA AAATTGCATG GCTTGCCGCT ATGCCCGCAC CCGGTTTACC 13741AACTTCATCG TGGACGACCG GGGAAGAATC CATCGATGGA AGTCTTCAAT AGTGGTGGAG 13801AAATTGGGCA AAGCTGAAGT CGGTGGTGAC CTTGTCAACA TTAAGCATGT TGTCCTCGAA 13861GGGGTTAAAG CTCAACCTTT GACGAGGACT TCGGCTGAGC AATGGGAAGC CTAGACGACT 13921TTTGCAACGA TCCCACCGCC GCACAAAAAC TCGTGCTGGC CTTTAGCATC ACATATACAC 13981CCATAATGAT ATACGCCCTT AAGGTGTCAC GCGGCCGACT CCTGGGGCTG TTGCACATCT 14041TGATATTTCT GAATTGTTCC TTTACTTTTG GGTACATGAC ATATGTGCAT TTTCAATCCA 14101CCAACCGTGT CGCATTCACT CTGGGGGCTG TAGTCGCCCT TTTGTGGGGT GTTTACAGCC 14161TCACAGAGTC ATGGAAGTTC ATCACTTCCA GATGCAGATT GTGTTGCCTA GGCCGGCGAT 14221ACATTCTGGC CCCTGCCCAT CACGTAGAAA GTGCTGCAGG CCTCCATTCA ATCCCAGCGT 14281CTGGTAACCG AGCATACGCT GTGAGAAAGC CCGGACTAAC ATCAGTGAAC GGCACTCTAG 14341TACCTGGGCT TCGGAGCCTC GTGCTGGGCG GCAAACGAGC TGTTAAACGA GGAGTGGTTA 14401ACCTCGTCAA GTATGGCCGG TAAGAACCAG AGCCAGAAGA AAAGAAGAAA TGCAGCTCCG 14461ATGGGGAAAG GCCAGCCAGT CAATCAACTG TGCCAGTTGC TGGGTACAAT GATAAAGTCC 14521CAGCGCCAGC AATCTAGGGG AGGACAGGCC AAAAAGAAGA AGCCTGAGAA GCCACATTTT 14581CCCCTAGCTG CTGAAGATGA CATTCGGCAC CATCTCACCC AGGCCGAACG TTCCCTCTGC 14641TTGCAATCGA TCCAGACGGC TTTCAATCAA GGCGCAGGAA CTGCGTCGCT TTCATCCAGC 14701GGGAAGGTCA GTTTCCAGGT TGAGTTCATG CTGCCGGTTG CTCATACAGT GCGCCTGATT 14761CGCGTGACTT CTACATCCGC CAGTCAGGGT GCAAATTAAT TTGACAGTCA GGTGAATGGC 14821CGCGATTGAC GTGTGGCCTC TAASEQ ID NO: 2: ORF 1a OF 94881 MSV ENCODED BY SEQUENCE OF SEQ ID NO: 1BETWEEN NUCLEOTIDES 178 . . . 7227MSGMFSRCMCTPAARVFWNAGQVYCTRCLSARSLLSPELQDTDLGAVGLFHKPKDKLHWKVPIGIPQVECSPSGCCWLSTIFPLARMTSGNHNFLQRLVKVADVLYRDGCLTPRHLRELQVYERGCNWYPITGPVPGMAVYANSMHVSDQPFPGATHVLTNSPLPQRACRQPFCPFEEAHSSIYRWEKFVIFMDSSSDGRSRMMWTPESDDSTALEVLPPELEHQVKVLVRSFPAHHLVDLADWELTESPDNGFSFSTSHPCGYLVRDPAVSEGKCWLSCFLSQSAEVLSREAHLATAYGYQTKWGVPGKYIQRRLQVHGLRAVVDPDGPIHVEALSCPQSWIRHLTLNDDVTPGFVRLMSLRIVPNTEPTTHRIFRFGVHKWYGAAGKRARGKRAAKSEKDSASTLKVARPTSTSGIVTYSPPADGSCGWHALAAILNRMINNDFTSPLPRYNRPEDDWASDGDLAQAIQCLQLPAAIARNRACPNAKYLIKLNGVHWEVEVRPGMAPRSLSRECVVGVCSEGCVASPYPEDGLPKRALEALASAYRLPSDCVCDGIIDFLANPPPQEFWTLDKMLTSPSPEQSGFSSLYKLLLEILPQKCGSTEGEFIYTVERMLKDCPSSKQAMALLAKIKVPSSKAPSVTLNECFPTDVPVNSELISWEEPKDPGAAVVLCPSDAKESKETAPEEAQARNRKVLHPVVLTEELSEQQVQVVEGDQDMPLDLTWPTLTATATPVRGPVPDNLSSGIGAQPATVQELILARPAPRLVERCGTESNGSSSFLDLPDVQTSDQPLDLSLAAWPVRATASDPGWIHGRREPVFVKPRGVFSDGESALQFGELSEASSVVDDRTKEAPVVDAPIDLTTSNETLSGSDPFEFAKFRRPRFSAQALIDRGGPLADVHAKIKSRVYEQCLQACEPGSRATPATKKWLDKMWDRVDMKTWRCTSQFQAGHILESLKFLPDMIQDTPPPVPRKNRAGDSAGLKQLVAQWDRKSSVTPPTKPVGPVLDQAVPLPMDIQQGDAISADKPPHSQNPSSQVDVGGGWKSFMLSGTRFAGSVSQRLTTWVFEVLSHLPAFMLTLFSPRGSMAPGDWLFAGAVLLALLLCRSYPILGCLPLLGVFSGSVRCVRLGVFGSWMAFAVFLFSTPPDPVGSSCDHDSPECHAELLALEQRQLWEPVRSLVVGPSGLLCVILGKLLGGSRCLWFVLLRICMLADLAISLIYVVSQGRCHKCWGKCIRTAPAEVALNVFPFSRATRSSLVSLCDRFQAPKGVDPVHLATGWRGCWCGESPIHQSHQKPIAYANLDEKKISAQTVIAVPYDPSQAIKCLKVLQAGGAIVDQPTPEVVRVSEIPFSAPFFPKVPVNPDCRVVVDSDTFVAAVRCGYSTAQLVLGRGNFAKLNQTPLRNSVPTKTTGGASYTLAVAQVSVWTLVHFILGLWLTSPQVCGRGTSDPWCSNPFSYPTYGPGVVCSSRLCVSADGVTLPLFSAVAHLSGREVGIFILVLASLGALAHRLALKADMSMVFLAFCAYAWPMSSWLICFFPMLLRWVTLHPLTMLWVHSFLVFCLPAAGVLSLGITGLLWAVGRFTQVAGIITPYDIHQYTSGPRGAAAVATAPEGTYMAAVRRAALTGRTLIFTPSAVGSLLEGAFRTQKPCLNTVNVVGSSLGSGGVFTIDGRRVIVTATHVLNGNTARVTGDSYNRMHTFNTNGDYAWSHADDWQGVAPMVKIAKGYRGRAYWQTSTGVEPGIMGEGFAFCFTNCGDSGSPVISEAGDLIGVHTGSNKLGSGLVTTPEGETCSIKETRLSDLSRHFAGPSVPLGDIKLSPAIIPDVTTIPSDLASLLASVPVMEGGLSTVQLLCVFFLLWRMMGHAWTPIVAVGFFLLNEILPAVLVRAVFSFALFVLAWATPWSAQVLMIRLLTAALNRNRLSLAFYAFGGVVGLATEIGTFAGGWPELSQALSTYCFLPRFLAVTSYVPTIIIGGLHALGVILWLFKYRCLHNMLVGDGSFSSAFFLRYFAEGNLRKGVSQSCGMNNESLTAALACKLSQADLDFLSSLTNFKCFVSASNMKNAAGQYIEAAYARALRQELASLVQVDKMKGVLAKLEAFAETATPSLDTGDVIVLLGQHPHGSILDINVGGERKTVSVQETRCLGGSKFSVCTVVSNTPVDTLTGIPLQTPTPLFENGPRHRSEDDDLKVERMKKHCVSLGFHKINGKVYCKIWDKSNGDTFYTDDSRYTQDHAFQDRSTDYRDRDYEGVQTAPQQGFDPKSEAPVGTVVIGGITYNRHLVKGKEVLVPKPDNCLEAARLSLEQALAGMGQTCDLTATEVEKLKRIISQLQGLTTEQALNCSEQ ID NO: 3 ORF 1B OF 94881 MSV ENCODED BY SEQUENCE OF SEQ ID NO: 1BETWEEN NUCLEOTIDES 7209 . . . 11600TGFKLLAASGLTRCGRGGLVVTETAVKIVKYHSRTFTLGSLDLKVTSEVEVKKSTEQGHAVVANLCSGVVLMRPHPPSLVDVLLKPGLDTTPGIQPGHGAGNMGVNGSIWDFETAPTKVELELSKQIIQACEVRRGDAPNLQLPYKLYPVRGDPERRKGRLVNTRFGDLPYKTPQDTKSAIHAACCLHPNGVLVSDGKSTLGTTLQHGFELYVPTVPYSVMEYLDSRPDTPFMCTKHGTSKAAAEDLQKYDLSTQGFVLPGVLRLVRRFIFSHVGKAPPLFLPSTYPAKNSMAGVNGQRFPTKDVQSIPEIDEMCARAVKENWQTVTPCTLKKQYCSKPKTRTILGTNNFIALAHRSALSGVTQAFMKKAWKSPIALGKNKFKELHCTVAGRCLEADLASCDRSTPAIVRWFVANLLYELAGCEEYLPSYVLNCCHDLVATQDGAFTKRGGLSSGDPVTSVSNTVYSLIIYAQHMVLSALKMGHEIGLKFLEEQLKFEDLLEIQPMLVYSDDLVLYAERPTFPNYHWWVEHLDLMLGFKTDPKKTVITDKPSFLGCRIEAGRQLVPNRDRILAALAYHMKAQNASEYYASAAAILMDSCACIDHDPEWYEDLICGIARCARQDGYRFPGPAFFMSMWEKLKSHNEGKKCRHCGICDAKADYASACGLDLCLFHSHFHQHCPVTLSCGHHAGSKECSQCQSPVGAGKSPLDAVLKQIPYKPPRTIIMKVDNKTTTLDPGRYQSRRGLVAVKRGIAGNEVDLSDGDYQVVPLLPTCKDINMVKVACNVLLSKFIVGPPGSGKTTWLLNQVQDDDVIYTPTHQTMFDIVSALKVCRYSIPGASGLPFPPPARSGPWVRLIASGHVPGRVSYLDEAGYCNHLDILRLLSKTPLVCLGDLQQLHPVGFDSYCYVFDQMPQKQLTTIYRFGPNICAAIQPCYREKLESKARNTRVVFTTRPVAFGQVLTPYHKDRTGSAITIDSSQGATFDIVTLHLPSPKSLNKSRALVAITRARHGLFIYDPHDQLQEFFNLTPERTDCNLAFSRGDELVVLNVDNAVTTVAKALETGSPRFRVSDPRCKSLLAACSASLEGSCMPLPQVAHNLGFYFSPDSPAFAPLPKELAPHWPVVTHQNNRAWPDRLVASMRPIDARYSKPMVGAGYVVGPSIFLGTPGVVSYYLTLYIGGEPQALPETLVSTGRIATDCREYLDAAEEEAARELPHAFIGDVKGTTIGGCHHITSKYLPRSLPKDSVAVVGVSSPGRAAKAVCTLTDVYLPELRPYLQPETASKCWKLKLDFRDVRLMVWKGATAYFQLEGLTWSALPDYARFIQLPKDAVVYIDPCIGPATANRKVVRTTDWRADLAVTPYDYGAQVILTTAWFEDLGPQWKILGLQPFRRTFGFENTEDWAILARRMNDGKDYTDYNWHCVRERPHAIYGRARDHTYHFALGTELQVELGRPR LPPEQVPSEQ ID NO: 4 ORF 2 OF 94881 MSV ENCODED BY SEQUENCE OF SEQ ID NO: 1BETWEEN NUCLEOTIDES 11611 . . . 12360MQWVYCGVKSVSCSWMPSLSSLLVWLTLSSFSPYCLGSLLQAGYWSSFSEWFAPRFSVRALPFTLPNYRRSYEGLLPNCRPDVPQFAVKHPLGILWHMRVSHLIDEMVSRRIYRTMEHSGQAAWKQVVSEATLTKLSRLDVVTHFQHLAAVEADSCRFLSSRLAMLKNLAVGNVSLEYNTTLDRVELIFPTPGTRPKLTDFRQWLISVHASIFSSVASSVTLFTVLWLRIPALRYVFGFHWPTATHHSNSEQ ID NO: 5 ORF 3 OF 94881 MSV ENCODED BY SEQUENCE OF SEQ ID NO: 1BETWEEN NUCLEOTIDES 12219 . . . 13016MAYQRARFHLLLCGFVCYLVHSALASNSSSTLCFWFPLAHGNTSFELTINYTICKPCPTSQAAQQRLEPGRNVWCKIGHDRCEERDHDELSMSIPSGYDNLKLEGYYAWLAFLSFSYAAQFHPELFGIGNVSRVFVDKRHQFICAEHDGQNSTISARHNISASYAVYYHHQIDGGNWFHLEWLRPFFSSWLVLNISWFLRRSPASPASRRIYQILRPTRPRLPVSWSFRTSIVSNLTGPQQRKVPLPSGGRPNVVKPSAFPSTSRSEQ ID NO: 6 ORF 4 OF 94881 MSV ENCODED BY SEQUENCE OF SEQ ID NO: 1BETWEEN NUCLEOTIDES 12761 . . . 13312MAATILFLLAGAQHLMVSEAFACKPCFSTHLSDIKTNTTAAAGFMVLQNINCFQSHRASTAQGTTPLRRSSQCREAVGIPQYITITANVTDESYLYNADLLMLSACLFYASEMSEKGFKVIFGNISGVVSACVNFTDYVAHVTQHTQQHHLVIDHIRLLHFLTPSTMRWATTIACLLAI LLAVSEQ ID NO: 7 ORF 5 OF 94881 MSV ENCODED BY SEQUENCE OF SEQ ID NO: 1BETWEEN NUCLEOTIDES 13309 . . . 13914MKCSCKLGHFLTPHSCFWWLFLLCTGLSWSFVDGNDDSSTSQYIYNLTICELNGTEWLSGHFDWAVETFVLYPVATHIISLGFLTTSHFLDALGLGAVSATGFIGERYVLSSMYGVCAFAAFVCFVIRAAKNCMACRYARTRFTNFIVDDRGRIHRWKSSIVVEKLGKAEVGGDLVNIKHVVLEGVKAQPLTRTSAEQWEASEQ ID NO: 8 ORF 6 OF 94881 MSV ENCODED BY SEQUENCE OF SEQ ID NO: 1BETWEEN NUCLEOTIDES 13902 . . . 14423MGSLDDFCNDPTAAQKLVLAFSITYTPIMIYALKVSRGRLLGLLHILIFLNCSFTFGYMTYVHFQSTNRVAFTLGAVVALLWGVYSLTESWKFITSRCRLCCLGRRYILAPAHHVESAAGLHSIPASGNRAYAVRKPGLTSVNGTLVPGLRSLVLGGKRAVKRGVVNLVKYGRSEQ ID NO: 9 ORF 7 OF 94881 MSV ENCODED BY SEQUENCE OF SEQ ID NO: 1BETWEEN NUCLEOTIDES 14413 . . . 14799MAGKNQSQKKRRNAAPMGKGQPVNQLCQLLGTMIKSQRQQSRGGQAKKKKPEKPHFPLAAEDDIRHHLTQAERSLCLQSIQTAFNQGAGTASLSSSGKVSFQVEFMLPVAHTVRLIRVTSTSASQGANSEQ ID NO: 10 FULL LENGTH NUCLEOTIDE SEQUENCE OF PARENT PRRSSTRAIN 94881 1TTTGTGTACC TTGGAGGCGT GGGTACAGCC CTGCCCCACC CCTTGGCCCC TGTTCTAGCC 61CGACAGGTAC CCTTCTCTCT CGGGGCGAGC GCGCCGCCTG CTGCTCCCTT GCGGCGGGAA 121GGACCTCCCG AGTATTTCCG GAGAGCACCT GCTTTACGGG ATCTCCGCCC TTTAACCATG 181TCTGGGATGT TCTCCCGGTG CATGTGCACC CCGGCTGCCC GGGTATTTTG GAACGCCGGC 241CAAGTCTATT GCACACGGTG TCTCAGTGCA CGGTCTCTTC TCTCTCCAGA ACTTCAGGAC 301ACGGACCTCG GTGCAGTTGG CTTGTTTCAC AAGCCTAAAG ACAAGCTCCA TTGGAAAGTT 361CCCATTGGTA TCCCCCAGGT GGAATGTTCT CCATCTGGGT GTTGCTGGCT GTCAACCATT 421TTTCCTTTAG CGCGCATGAC CTCCGGCAAT CACAACTTCC TTCAACGACT CGTGAAGGTT 481GCTGACGTAT TGTACCGTGA CGGTTGCTTA ACCCCTAGAC ACCTCCGTGA ACTCCAAGTT 541TACGAGCGTG GTTGCAATTG GTATCCGATT ACGGGGCCTG TGCCTGGGAT GGCTGTGTAC 601GCGAACTCCA TGCACGTGTC CGACCAACCG TTCCCTGGTG CCACTCATGT GTTAACAAAT 661TCCCCTTTGC CTCAACGGGC TTGTCGGCAG CCGTTCTGTC CGTTCGAAGA GGCCCATTCT 721AGCATATACA GGTGGGAAAA ATTTGTAATT TTTATGGATT CCTCCTCCGA CGGTCGATCT 781CGCATGATGT GGACTCCGGA ATCCGATGAC TCCACGGCTT TGGAAGTTCT GCCGCCCGAG 841CTAGAACACC AGGTCAAGGT CCTTGTTCGG AGCTTTCCCG CCCATCACCT TGTCGACCTT 901GCCGATTGGG AGCTCACTGA GTCCCCTGAG AACGGTTTTT CCTTCAGCAC GTCACATCCT 961TGCGGCTACC TTGTTCGGGA CCCGGCTGTA TCCGAAGGCA AGTGTTGGCT TTCCTGCTTT 1021TTGAGCCAGT CAGCCGAAGT GCTCAGTCGC GAGGCGCATC TGGCTACCGC CTATGGTTAC 1081CAAACCAAGT GGGGTGTGCC TGGCAAGTAC ATCCAGCGCA GACTTCAAGT TCACGGTCTC 1141CGTGCTGTGG TCGACCCTGA TGGTCCCATT CACGTTGAAG CATTGTCTTG CCCCCAGTCT 1201TGGATCAGGC ACTTGACCCT GAATGATGAT GTCACCCCGG GATTCGTTCG CCTAATGTCT 1261CTTCGCATTG TGCCGAACAC AGAGCCTACC ACACACCGGA TCTTTCGTTT TGGAGTGCAC 1321AAGTGGTATG GTGCCGCCGG CAAACGGGCC CGTGGCAAGC GTGCCGCCAA AAGTGAGAAA 1381GACTCGGCTT CCACCCTCAA GGTTGCCCGA CCGACTTCCA CCAGTGGAAT CGTCACCTAC 1441TCCCCACCTG CGGACGGGTC TTGTGGTTGG CATGCCCTTG CCGCCATACT GAACCGGATG 1501ATTAATAATG ACTTCACGTC CCCTCTGCCT CGGTACAACA GGCCGGAGGA CGATTGGGCT 1561TCTGATGGTG ACCTTGCTCA GGCCATTCAA TGTTTGCAAC TACCTGCCGC CATAGCTCGG 1621AACCGCGCCT GCCCTAACGC CAAATACCTC GTAAAACTCA ACGGAGTTCA TTGGGAGGTA 1681GAGGTGAGGC CTGGAATGGC TCCTCGCTCC CTCTCTCGTG AGTGCGTTGT TGGCGTCTGC 1741TCTGAAGGCT GTGTCGCGTC GCCTTACCCG GAGGACGGGT TGCCTAAACG TGCACTTGAG 1801GCCCTGGCGT CTGCTTATAG ACTGCCTTCA GACTGTGTTT GTGATGGTAT TATTGACTTC 1861CTTGCCAATC CACCTCCCCA GGAGTTCTGG ACTCTTGACA AAATGTTGAC TTCCCCGTCA 1921CCGGAGCAGT CCGGCTTCTC TAGTCTGTAT AAATTGTTGT TAGAGGTCTT GCCGCAGAAA 1981TGCGGATCCA CAGAAGGGGA ATTCATCTAT ACTGTTGAGA GGATGTTGAA GGATTGTCCG 2041AGCTCCAAAC AGGCCATGGC CCTCCTTGCA AAAATTAAGG TCCCATCCTC AAAGGCCCCA 2101TCCGTGACTC TGAACGAGTG CTTCCCCACG GATGTTCCAG TCAACTCTGA GTTAATATCT 2161TGGGAAGAGC CCAAAGACCC TGGCGCTGCT GTTGTCCTAT GTCCATCGGA TGCAAAAGAA 2221TCTAAGGAAA CAGCCCCTGA AGAAGCTCAA GCGAGAAACC GTAAGGTCCT CCACCCTGTG 2281GTCCTTACCG AGGAACTTAG CGAGCAACAG GTGCAGGTGG TTGAGGGTGA TCAGGATATG 2341CCACTGGATT TGACTTGGCC AACCTTAACC GCTACGGCGA CCCCTGTTAG AGGGCCGGTA 2401CCGGACAATT TGAGCTCTGG CATTGGTGCC CAGCCCGCTA CCGTTCAAGA ACTCATTCTG 2461GCGAGGCCTG CACCCCGTCT TGTTGAGCGC TGTGGCACGG AGTCGAACGG CAGCAGTTCA 2521TTTCTGGATT TGCCTGACGT GCAGACCTCG GACCAGCCTT TAGACCTGTC CCTGGCCGCG 2581TGGCCTGTAA GGGCTACCGC GTCTGACCCC GGTTGGATCC ACGGTAGGCG TGAGCCTGTC 2641TTTGTGAAGC CTCGAGGTGT TTTCTCTGAT GGCGAGTCGG CCCTTCAGTT CGGAGAGCTT 2701TCCGAAGCCA GTTCTGTCGT CGATGACCGG ACAAAAGAAG CTCCGGTGGT TGACGCCCCC 2761ATCGATTTGA CAACTTCGAA CGAGACGCTC TCTGGGTCTG ACCCCTTTGA ATTCGCCAAA 2821TTCAGGCGCC CGCGTTTCTC CGCGCAAGCT TTAATCGACC GAGGTGGTCC GCTTGCCGAT 2881GTTCATGCAA AGATAAAGAG TCGGGTATAT GAACAATGCC TTCAAGCTTG TGAACCTGGT 2941AGTCGTGCGA CCCCAGCCAC CAAGAAGTGG CTCGACAAAA TGTGGGACAG GGTGGACATG 3001AAAACTTGGC GCTGCACCTC GCAGTTCCAA GCTGGTCACA TTCTTGAGTC CCTCAAATTC 3061CTCCCTGACA TGATTCAAGA CACACCGCCT CCTGTTCCCA GGAAGAACCG AGCTGGTGAC 3121AGTGCCGGCC TGAAGCAACT GGTGGCGCAG TGGGATAGGA AATTGAGTGT GACACCCCCC 3181ACAAAACCGG TTGGACCGGT GCTTGACCAG ACCGTCCCTC TGCCTATGGA CATCCAGCAA 3241GAAGATGCCA TCTCCGCTGA CAAGCCACCC CATTCGCAAA ACCCTTCTAG TCAAGTAGAT 3301GTGGGTGGAG GTTGGAAAAG TTTTATGCTC TCCGGCACCC GTTTCGCGGG GTCCGTTAGT 3361CAGCGCCTTA CGACATGGGT TTTTGAGGTT CTCTCCCATC TCCCAGCTTT TATGCTCACA 3421CTTTTCTCGC CACGGGGCTC TATGGCTCCA GGTGATTGGC TGTTTGCAGG TGCTGTTCTA 3481CTTGCTCTCC TGCTCTGCCG TTCTTACCCA ATACTCGGAT GCCTTCCCTT ATTGGGTGTC 3541TTTTCTGGTT CTGTGCGGTG TGTTCGTTTG GGTGTTTTTG GTTCTTGGAT GGCTTTTGCT 3601GTATTTTTAT TCTCGACTCC ACCCGACCCA GTCGGTTCTT CTTGTGACCA CGATTCGCCG 3661GAGTGTCATG CTGAGCTTTT GGCTCTTGAG CAGCGCCAAC TTTGGGAACC TGTGCGCAGC 3721CTTGTGGTCG GGCCATCGGG CCTCTTATGC GTCATTCTTG GCAAGTTACT CGGTGGGTCA 3781CGTTGTCTCT GGTTTGTTCT CCTACGTATA TGCATGCTCG CAGATTTGGC AATTTCTCTT 3841ATTTATGTGG TGTCCCAAGG GCGTTGTCAC AAGTGTTGGG GAAAGTGTAT AAGGACGGCT 3901CCTGCAGAAG TGACCCTTAA TGTGTTTCCT TTTTCGCGCG CCACCCGCTC ATCTCTTGTG 3961TCCTTGTGTG ATCGGTTCCA AGCGCCAAAA GGAGTTGACC CCGTGCACTT GGCGACAGGC 4021TGGCGCGGGT GCTGGTGTGG TGAGAGCCCT ATTCATCAAT CACACCAAAA ACCGATAGCT 4081TATGCCAACT TGGATGAAAA GAAGATATCC GCCCAGACGG TGATTGCTGT CCCGTATGAT 4141CCCAGTCAGG CCATTAAATG CCTGAAAGTT TTGCAGGCAG GAGGGGCTAT TGTGGACCAG 4201CCTACGCCCG AGGTCGTCCG TGTGTCTGAG ATTCCCTTCT CGGCCCCATT TTTTCCGAAG 4261GTCCCAGTCA ACCCAGATTG CAGGGTTGTG GTAGATTCGG ACACTTTTGT GGCTGCGGTC 4321CGCTGCGGTT ATTCGACAGC ACAACTGGTC CTTGGTCGGG GCAACTTTGC CAAGCTAAAT 4381CAGACCCCCC TCAGGAACTC TGTCCCCACC AAAACAACTG GTGGGGCCTC ATACACCCTT 4441GCCGTGGCCC AGGTATCTGT GTGGACTCTT GTTCATTTCA TCCTCGGCCT TTGGTTAACG 4501TCACCTCAAG TGTGTGGTCG AGGGACCTCT GACCCGTGGT GTTCGAACCC TTTTTCGTAT 4561CCTACTTATG GCCCCGGAGT TGTGTGTTCC TCTCGACTCT GCGTGTCTGC CGACGGAGTT 4621ACCCTGCCAT TGTTCTCAGC CGTTGCCCAT CTTTCCGGTA GAGAGGTGGG GATTTTTATT 4681TTGGTGCTTG CCTCCTTGGG CGCTTTAGCC CACCGCTTGG CTCTTAAGGC AGACATGTCA 4741ATGGTCTTTT TGGCGTTTTG TGCTTACGCC TGGCCCATGA GCTCCTGGTT AATTTGCTTC 4801TTTCCTATGC TCTTGAGGTG GGTAACCCTT CATCCTCTCA CTATGCTTTG GGTGCACTCA 4861TTTTTGGTGT TTTGCCTACC AGCTGCCGGC GTTCTCTCGC TGGGAATAAC CGGTCTTCTT 4921TGGGCAGTTG GCCGTTTCAC CCAGGTTGCC GGAATTATCA CACCTTATGA CATCCACCAG 4981TATACCTCCG GACCACGTGG TGCAGCTGCT GTAGCAACGG CTCCAGAAGG TACTTACATG 5041GCGGCCGTTC GGAGAGCCGC TTTGACTGGA CGGACTTTGA TCTTCACACC ATCTGCAGTC 5101GGATCCCTTC TTGAAGGTGC TTTCAGAACT CAAAAGCCCT GCCTTAACAC CGTGAATGTC 5161GTAGGCTCTT CCCTTGGTTC TGGAGGAGTT TTCACCATTG ATGGCAGAAG AGTCATCGTC 5221ACTGCCACCC ATGTGTTGAA TGGTAACACA GCCAGGGTCA CTGGTGATTC CTACAACCGC 5281ATGCACACGT TCAATACTAA TGGTGATTAT GCCTGGTCCC ATGCTGATGA CTGGCAAGGC 5341GTTGCCCCTA TGGTTAAGAT CGCTAAGGGG TATCGCGGTC GTGCCTACTG GCAAACGTCA 5401ACCGGAGTCG AACCTGGCAT CATGGGGGAA GGATTCGCCT TCTGTTTCAC TAACTGTGGC 5461GACTCAGGGT CACCTGTCAT TTCAGAAGCT GGTGACCTTA TTGGAGTCCA TACCGGTTCA 5521AACAAACTCG GTTCTGGTCT TGTGACAACC CCTGAAGGGG AGACCTGCTC CATCAAGGAA 5581ACTAGGCTCT CTGACCTTTC TAGACATTTT GCAGGTCCAA GCGTCCCTCT TGGGGACATT 5641AAGTTGAGCC CAGCCATCAT CCCTGATGTG ACAACTATTC CGAGTGACTT GGCATCGCTC 5701CTTGCTTCTG TCCCCGTGAT GGAAGGTGGC CTCTCAACTG TCCAGCTTTT GTGCGTCTTT 5761TTCCTTCTCT GGCGCATGAT GGGCCATGCC TGGACACCCA TTGTTGCCGT AGGCTTCTTT 5821TTGCTGAATG AAATTCTCCC AGCAGTCTTG GTCCGAGCTG TGTTCTCTTT TGCACTCTTT 5881GTACTTGCAT GGGCCACCCC CTGGTCGGCA CAAGTGTTGA TGATTAGACT CCTCACGGCG 5941GCTCTCAACC GCAACAGGTT GTCCCTGGCG TTCTACGCAC TCGGAGGTGT CGTTGGCCTG 6001GCCACAGAAA TCGGGACTTT TGCTGGTGGA TGGCCTGAAC TGTCCCAAGC CCTCTCGACA 6061TACTGCTTCC TGCCCAGGTT CCTTGCTGTG ACTAGTTATG TCCCCACCAT CATCATCGGT 6121GGGCTCCATG CCCTCGGCGT AATTTTGTGG TTATTCAAAT ACCGATGCCT CCACAACATG 6181CTGGTTGGTG ATGGGAGTTT CTCAAGCGCT TTCTTCCTAC GGTATTTTGC TGAGGGTAAT 6241CTTAGGAAAG GCGTGTCGCA GTCCTGTGGC ATGAATAACG AATCCCTGAC AGCTGCTTTG 6301GCTTGCAAGT TGTCGCAAGC TGACCTTGAT TTTTTGTCCA GTTTAACGAA CTTCAAGTGC 6361TTTGTGTCCG CTTCAAACAT GAAAAATGCA GCTGGCCAAT ACATCGAGGC GGCGTATGCT 6421AGAGCTCTGC GTCAGGAGCT GGCCTCCTTG GTTCAGGTTG ACAAGATGAA AGGAGTATTG 6481GCCAAGCTCG AGGCTTTCGC TGAGACGGCC ACTCCGTCAC TTGACACAGG TGACGTGATT 6541GTTCTGCTTG GGCAACACCC CCATGGATCC ATCCTCGACA TTAATGTGGG GGGTGAAAGG 6601AAAACTGTGT CTGTGCAAGA AACACGATGC CTGGGTGGTT CCAAATTCAG TGTCTGCACT 6661GTCGTGTCCA ACACGCCCGT GGATACCTTG ACCGGCATCC CACTTCAGAC GCCAACCCCA 6721CTTTTTGAAA ATGGCCCGCG CCATCGCAGC GAGGACGACG ACCTTAAAGT TGAGAGAATG 6781AAAAAACACT GTGTATCCCT CGGCTTCCAC AAAATCAATG GTAAAGTTTA CTGCAAAATT 6841TGGGACAAGT CTAACGGCGA CACCTTTTAC ACGGATGATT CCCGATACAC TCAAGACCAT 6901GCTTTTCAGG ACAGGTCAAC CGACTATAGA GACAGGGATT ATGAAGGTGT ACAGACCGCC 6961CCCCAACAGG GATTCGATCC AAAGTCCGAA GCCCCTGTTG GCACTGTTGT AATCGGTGGC 7021ATTACGTATA ACAGGCATCT GGTCAAAGGT AAGGAGGTCC TAGTTCCCAA ACCTGACAAC 7081TGCCTTGAAG CTGCCAGACT GTCCCTTGAG CAAGCTCTTG CTGGGATGGG CCAAACTTGT 7141GACCTTACAG CTACCGAAGT GGAGAAACTA AAGCGCATCA TTAGTCAACT CCAAGGTCTG 7201ACCACTGAAC AGGCTTTAAA CTGCTAGCCG CCAGCGGCTT GACCCGCTGT GGCCGCGGCG 7261GCCTAGTTGT AACTGAAACG GCGGTAAAAA TCGTAAAATA CCACAGCAGA ACTTTCACCT 7321TAGGCTCTTT AGACCTAAAA GTCACCTCCG AGGTGGAGGT GAAGAAATCA ACTGAGCAGG 7381GGCACGCTGT CGTGGCGAAC TTATGTTCCG GTGTCGTCTT GATGAGGCCT CACCCACCGT 7441CCCTTGTTGA CGTTCTCCTC AAACCCGGAC TTGACACAAC ACCCGGCATT CAACCAGGGC 7501ATGGGGCCGG GAATATGGGC GTGAACGGTT CTATTTGGGA TTTTGAAACT GCACCCACAA 7561AGGTAGAACT AGAGTTGTCC AAGCAAATAA TCCAAGCATG TGAAGTCAGG CGCGGGGACG 7621CCCCTAACCT CCAACTCCCC TACAAGCTTT ATCCTGTCAG GGGGGACCCC GAGCGGCGTA 7681AAGGTCGCCT TGTCAACACT AGGTTTGGAG ATTTACCTTA CAAAACTCCC CAAGACACCA 7741AGTCCGCAAT TCATGCGGCT TGTTGCCTGC ATCCCAATGG GGTCCTCGTG TCTGATGGTA 7801AATCCACGCT GGGTACCACT CTTCAACATG GTTTCGAGCT TTATGTCCCC ACTGTACCTT 7861ATAGTGTCAT GGAATACCTT GATTCACGCC CTGACACCCC TTTTATGTGT ACTAAACATG 7921GCACTTCCAA GGCTGCTGCA GAGGACCTCC AAAAATATGA CCTATCCACT CAAGGGTTTG 7981TCTTGCCTGG GGTCCTACGC CTAGTGCGCA GGTTCATCTT TAGCCATGTT GGTAAGGCGC 8041CACCACTGTT CCTTCCATCA ACCTACCCTG CCAAGAACTC CATGGCAGGG GTCAATGGCC 8101AGAGGTTCCC AACAAAGGAT GTCCAGAGCA TACCTGAAAT TGATGAAATG TGCGCCCGTG 8161CCGTCAAGGA AAATTGGCAG ACTGTGACAC CTTGCACCCT CAAAAAACAG TACTGTTCCA 8221AACCTAAAAC TAGAACCATC CTAGGTACCA ACAACTTCAT AGCCTTGGCT CACAGGTCAG 8281CACTCAGTGG TGTCACCCAG GCGTTCATGA AGAAGGCCTG GAAGTCCCCA ATTGCCTTGG 8341GGAAAAACAA GTTTAAGGAA TTGCATTGCA CTGTCGCCGG CAGATGCCTT GAGGCTGACC 8401TGGCTTCCTG CGATCGCAGC ACCCCCGCCA TTGTGAGGTG GTTTGTTGCC AACCTCCTGT 8461ATGAACTTGC AGGATGTGAA GAGTACTTGC CTAGCTACGT GCTCAACTGT TGCCATGACC 8521TTGTGGCAAC GCAGGATGGC GCTTTCACAA AACGCGGTGG CCTGTCGTCC GGGGACCCCG 8581TCACCAGTGT GTCCAACACC GTCTACTCAC TGATAATTTA CGCCCAGCAC ATGGTGCTTT 8641CGGCCTTGAA GATGGGTCAT GAAATTGGTC TCAAGTTCCT TGAGGAACAG CTCAAATTTG 8701AGGACCTTCT TGAAATCCAG CCCATGTTAG TGTATTCTGA TGACCTCGTC TTGTATGCGG 8761AAAGACCCAC TTTTCCCAAC TACCATTGGT GGGTCGAGCA TCTTGACCTG ATGTTGGGCT 8821TTAAAACGGA CCCAAAGAAA ACTGTCATAA CTGATAAACC CAGTTTTCTC GGCTGCAGAA 8881TTGAAGCAGG ACGGCAGTTA GTCCCCAATC GCGACCGTAT TCTGGCTGCT CTTGCATATC 8941ATATGAAGGC GCAGAACGCC TCAGAGTATT ATGCGTCCGC TGCCGCAATT CTGATGGATT 9001CGTGTGCTTG CATTGACCAT GACCCCGAGT GGTATGAGGA CCTTATCTGC GGCATCGCCC 9061GGTGTGCTCG CCAGGACGGT TACCGTTTTC CAGGCCCGGC ATTTTTCATG TCCATGTGGG 9121AGAAGCTGAA AAGTCATAAC GAAGGGAAGA AATGCCGTCA CTGCGGCATC TGCGACGCCA 9181AAGCCGACTA TGCGTCCGCC TGTGGACTTG ATTTGTGTTT GTTCCATTCA CACTTTCATC 9241AACACTGCCC AGTCACTCTG AGCTGTGGCC ACCATGCCGG TTCAAAGGAA TGTTCGCAGT 9301GTCAGTCACC TGTCGGGGCT GGCAAATCCC CCCTTGACGC TGTGCTGAAA CAAATCCCGT 9361ACAAACCTCC TCGTACCATT ATCATGAAGG TGGACAACAA AACAACGACC CTTGACCCGG 9421GAAGATATCA GTCCCGTCGA GGTCTTGTTG CAGTCAAAAG AGGTATTGCA GGTAATGAGG 9481TTGATCTTTC TGATGGAGAC TACCAAGTGG TGCCTCTTTT GCCGACTTGC AAAGACATAA 9541ACATGGTGAA GGTGGCTTGC AACGTACTAC TCAGCAAGTT TATAGTAGGG CCGCCAGGTT 9601CCGGAAAAAC CACCTGGCTA CTGAACCAAG TCCAGGACGA TGATGTCATT TACACACCTA 9661CTCATCAGAC AATGTTTGAC ATAGTCAGTG CTCTTAAAGT TTGCAGGTAT TCCATCCCAG 9721GAGCCTCAGG ACTCCCTTTT CCACCACCTG CCAGGTCCGG GCCGTGGGTT AGGCTCATCG 9781CCAGCGGACA TGTCCCTGGC CGAGTGTCAT ATCTCGATGA GGCAGGATAT TGCAATCATC 9841TAGACATTCT AAGGCTGCTT TCCAAAACAC CCCTTGTGTG TTTGGGTGAC CTTCAGCAAC 9901TTCACCCGGT CGGCTTTGAT TCCTATTGTT ATGTGTTCGA TCAGATGCCT CAGAAGCAGC 9961TGACCACCAT TTATAGATTT GGCCCTAACA TCTGTGCAGC CATCCAGCCT TGTTACAGGG 10021AGAAACTTGA ATCCAAGGCC AGGAACACCA GAGTGGTTTT CACCACCCGG CCTGTGGCCT 10081TTGGTCAGGT CCTGACACCG TACCACAAAG ATCGTACCGG CTCTGCAATA ACTATAGATT 10141CATCCCAGGG GGCGACCTTC GACATTGTGA CATTGCATCT ACCATCGCCA AAGTCCCTAA 10201ACAAATCCCG AGCACTTGTA GCCATCACTC GGGCAAGACA TGGGTTGTTC ATTTATGACC 10261CTCATGACCA ACTCCAGGAG TTTTTCAACT TAACCCCCGA GCGCACTGAT TGTAACCTTG 10321CGTTCAGCCG TGGGGATGAG CTGGTTGTTT TGAATGTGGA TAATGCGGTC ACAACTGTAG 10381CGAAGGCCCT AGAGACAGGT TCACCCCGAT TTCGAGTATC GGACCCGAGG TGCAAGTCTC 10441TCTTAGCCGC TTGTTCGGCC AGTCTAGAAG GGAGCTGCAT GCCACTACCA CAAGTAGCAC 10501ATAACCTGGG GTTTTACTTT TCCCCGGACA GCCCAGCTTT TGCACCCCTG CCAAAAGAGC 10561TGGCGCCACA TTGGCCAGTG GTCACCCACC AGAATAATCG AGCGTGGCCT GATCGACTTG 10621TCGCTAGTAT GCGCCCAATT GATGCCCGCT ACAGCAAGCC AATGGTCGGT GCAGGGTATG 10681TGGTCGGGCC ATCCATTTTT CTTGGCACTC CTGGTGTGGT GTCATACTAT CTCACATTAT 10741ACATCGGGGG CGAGCCTCAG GCCCTGCCAG AAACACTCGT TTCAACAGGA CGTATAGCCA 10801CAGATTGTCG GGAATATCTC GACGCGGCTG AGGAAGAGGC AGCGAGAGAA CTTCCCCACG 10861CATTTATTGG CGATGTCAAA GGCACTACGG TCGGGGGGTG TCACCACATT ACATCGAAAT 10921ACCTACCTAG GTCCCTGCCT AAAGACTCTG TTGCTGTGGT TGGGGTGAGT TCGCCCGGTA 10981GGGCTGCTAA AGCCGTGTGC ACTCTCACCG ATGTGTACCT CCCCGAACTC CGACCATATT 11041TGCAACCGGA GACGGCATCA AAATGCTGGA AACTTAAACT GGATTTCAGG GATGTTCGAC 11101TGATGGTCTG GAAAGGCGCC ACAGCCTATT TCCAGTTGGA AGGGCTGACA TGGTCAGCGC 11161TGCCCGATTA TGCTAGGTTC ATTCAGCTAC CCAAGGATGC CGTTGTGTAC ATCGATCCGT 11221GTATAGGGCC GGCAACAGCC AATCGCAAGG TTGTGCGAAC CACAGACTGG CGGGCCGACC 11281TGGCAGTGAC ACCGTATGAT TACGGTGCTC AGGTCATTTT GACAACAGCC TGGTTCGAGG 11341ACCTTGGGCC GCAGTGGAAG ATTTTGGGGT TGCAGCCTTT CAGACGAACA TTTGGCTTTG 11401AGAACACTGA AGATTGGGCA ATTCTCGCAC GCCGTATGAA TGACGGCAAA GATTACACTG 11461ACTATAATTG GCATTGTGTA CGAGAACGCC CACACGCAAT TTACGGGCGC GCCCGTGACC 11521ATACGTATCA TTTTGCCCTT GGCACTGAAC TGCAAGTAGA GCTGGGCAGA CCCCGGCTGC 11581CTCCTGAGCA AGTGCCGTGA ACGCGGAGTG ATGCAATGGG TTCACTGTGG AGTAAAATCA 11641GTCAGTTGTT CGTGGATGCC TTCACTGAGT TCCTTGTTAG TGTGGTTGAC ATTGTCATCT 11701TTCTCGCCAT ATTGTTTGGG TTCACTGTTG CAGGCTGGTT ATTGGTCTTC CTTCTCAGAG 11761TGGTTTGCTC CGCGTTTCTC CGTTCGCGCT CTGCCATTCA CTCTCCCGAA CTATCGAAGG 11821TCCTATGAGG GCTTGCTACC CAACTGCAGA CCGGATGTCC CACAATTCGC AGTTAAGCAC 11881CCGTTGGGTA TACTTTGGCA TATGCGAGTC TCCCACCTAA TTGACGAAAT GGTCTCTCGC 11941CGCATTTACC GGACCATGGA ACATTCGGGT CAAGCGGCCT GGAAGCAGGT TGTTAGTGAA 12001GCCACTCTCA CAAAACTGTC AAGGCTTGAC GTAGTCACTC ATTTCCAACA CCTGGCCGCA 12061GTGGAGGCTG ATTCTTGCCG CTTCCTTAGC TCACGACTCG CGATGCTGAA AAACCTTGCC 12121GTTGGCAATG TGAGCCTGGA GTACAACACT ACTTTGGACC GCGTTGAGCT CATCTTTCCC 12181ACACCAGGTA CGAGGCCCAA GTTGACCGAT TTTAGGCAAT GGCTTATCAG CGTGCACGCT 12241TCCATCTTCT CCTCTGTGGC TTCGTCTGTT ACCTTGTTCA CAGTGCTTTG GCTTCGAATT 12301CCAGCTCTAC GCTATGTTTT TGGTTTCCAT TGGCCCACGG CAACACATCA TTCGAACTAA 12361CTATCAATTA CACTATATGT AAGCCATGCC CTACCAGTCA AGCTGCCCAA CAAAGACTCG 12421AGCCTGGCCG TAACGTGTGG TGCAAAATAG GGCACGACAG GTGTGAGGAA CGTGACCATG 12481ATGAGTTGTC AATGTCCATT CCGTCCGGGT ACGACAACCT CAAACTTGAG GGTTATTATG 12541CTTGGCTGGC TTTTTTGTCC TTTTCCTACG CGGCCCAATT CCATCCGGAG CTGTTCGGAA 12601TAGGAAACGT GTCGCGCGTC TTTGTGGATA AGCGACACCA GTTCATTTGC GCCGAGCATG 12661ATGGACAAAA TTCAACCATA TCTGCCAGAC ACAACATCTC CGCGTCGTAT GCGGTGTATT 12721ACCATCATCA AATAGACGGG GGCAATTGGT TTCATTTGGA ATGGCTGCGA CCATTCTTTT 12781CCTCCTGGCT GGTGCTCAAC ATCTCATGGT TTCTGAGGCG TTCGCCTGCA AGCCCTGCTT 12841CTCGACGCAT CTATCAGATA TTAAGACCAA CACGACCGCG GCTGCCGGTT TCATGGTCCT 12901TCAGAACATC AATTGTTTCC AATCTCACAG GGCCTCAACA GCGCAAGGTA CCACTCCCCT 12961CAGGAGGTCG TCCCAATGTC GTGAAGCCGT CGGCATTCCC CAGTACATCA CGATAACGGC 13021TAATGTGACC GATGAATCGT ATTTGTACAA CGCGGACTTG CTGATGCTTT CCGCGTGCCT 13081TTTCTACGCC TCGGAAATGA GCGAGAAAGG CTTCAAAGTC ATCTTTGGGA ATATTTCTGG 13141CGTTGTTTCC GCTTGTGTTA ATTTCACAGA TTATGTGGCC CATGTGACCC AACACACTCA 13201GCAGCACCAT TTGGTAATTG ATCACATTCG GTTACTACAC TTCTTGACAC CGTCTACGAT 13261GAGGTGGGCT ACAACCATTG CTTGTTTGTT TGCCATTCTT TTGGCGGTAT GAAATGTTCT 13321TGCAAGTTGG GGCATTTCTT GACTCCTCAC TCTTGCTTCT GGTGGCTTTT TTTGCTGTGT 13381ACCGGCTTGT CTTGGTCCTT TGTCGATGGC AACGACAACA GCTCGACATC CCAATACATA 13441TATAATTTGA CGATATGCGA GCTGAATGGG ACCGAATGGT TGTCCGGTCA TTTTGATTGG 13501GCAGTCGAAA CCTTTGTGCT TTACCCAGTT GCCACTCATA TCATTTCACT GGGTTTTCTC 13561ACAACAAGCC ATTTCCTTGA TGCGCTCGGT CTCGGCGCTG TGTCCGCCAC AGGATTCATT 13621GGCGAGCGGT ATGTACTTAG CAGCATGTAC GGCGTTTGCG CCTTCGCGGC GCTCGTATGT 13681TTTGTCATCC GTGCTGCTAA AAATTGCATG GCTTGCCGCT ATGCCCGCAC CCGGTTTACC 13741AACTTCATCG TGGACGACCG GGGAAGAATC CATCGATGGA AGTCTTCAAT AGTGGTGGAG 13801AAATTGGGCA AAGCTGAAGT CGGTGGTGAC CTTGTCAACA TTAAGCATGT TGTCCTCGAA 13861GGGGTTAAAG CTCAACCCTT GACGAGGACT TCGGCTGAGC AATGGGAAGC CTAGACGACT 13921TTTGCAACGA TCCCACCGCC GCACAAAAAC TCGTGCTGGC CTTTAGCATC ACATATACAC 13981CCATAATGAT ATACGCCCTT AAGGTGTCAC GCGGCCGACT CCTGGGGCTG TTGCACATCT 14041TGATATTTCT GAATTGTTCC TTTACTTTTG GGTACATGAC ATATGTGCAT TTTCAATCCA 14101CCAACCGTGT CGCACTCACT CTGGGGGCTG TAGTCGCCCT TTTGTGGGGT GTTTACAGCC 14161TCACAGAGTC ATGGAAGTTC ATCACTTCCA GATGCAGATT GTGTTGCCTA GGCCGGCGAT 14221ACATTCTGGC CCCTGCCCAT CACGTAGAAA GTGCTGCAGG CCTCCATTCA ATCCCAGCGT 14281CTGGTAACCG AGCATACGCT GTGAGAAAGC CCGGACTAAC ATCAGTGAAC GGCACTCTAG 14341TACCTGGGCT TCGGAGCCTC GTGCTGGGCG GCAAACGAGC TGTTAAACGA GGAGTGGTTA 14401ACCTCGTCAA GTATGGCCGG TAAGAACCAG AGCCAGAAGA AAAGAAGAAA TGCAGCTCCG 14461ATGGGGAAAG GCCAGCCAGT CAATCAACTG TGCCAGTTGC TGGGTACAAT GATAAAGTCC 14521CAGCGCCAGC AATCTAGGGG AGGACAGGCC AAAAAGAAGA AGCCTGAGAA GCCACATTTT 14581CCCCTAGCTG CTGAAGATGA CATTCGGCAC CATCTCACCC AGGCCGAACG TTCCCTCTGC 14641TTGCAATCGA TCCAGACGGC TTTCAATCAA GGCGCAGGAA CTGCGTCGCT TTCATCCAGC 14701GGGAAGGTCA GTTTCCAGGT TGAGTTCATG CTGCCGGTTG CTCATACAGT GCGCCTGATT 14761CGCGTGACTT CTACATCCGC CAGTCAGGGT GCAAATTAAT TTGACAGTCA GGTGAATGGC 14821CGCGATTGAC GTGTGGCCTC TAASEQ ID NO: 11 ORF 1a OF PARENTAL PRRSV STRAIN 94881 ENCODED BYSEQUENCE OF SEQ ID NO: 10 BETWEEN NUCLEOTIDES 178 . . . 7227MSGMFSRCMCTPAARVFWNAGQVYCTRCLSARSLLSPELQDTDLGAVGLFHKPKDKLHWKVPIGIPQVECSPSGCCWLSTIFPLARMTSGNHNFLQRLVKVADVLYRDGCLTPRHLRELQVYERGCNWYPITGPVPGMAVYANSMHVSDQPFPGATHVLTNSPLPQRACRQPFCPFEEAHSSIYRWEKFVIFMDSSSDGRSRMMWTPESDDSTALEVLPPELEHQVKVLVRSFPAHHLVDLADWELTESPENGFSFSTSHPCGYLVRDPAVSEGKCWLSCFLSQSAEVLSREAHLATAYGYQTKWGVPGKYIQRRLQVHGLRAVVDPDGPIHVEALSCPQSWIRHLTLNDDVTPGFVRLMSLRIVPNTEPTTHRIFRFGVHKWYGAAGKRARGKRAAKSEKDSASTLKVARPTSTSGIVTYSPPADGSCGWHALAAILNRMINNDFTSPLPRYNRPEDDWASDGDLAQAIQCLQLPAAIARNRACPNAKYLVKLNGVHWEVEVRPGMAPRSLSRECVVGVCSEGCVASPYPEDGLPKRALEALASAYRLPSDCVCDGIIDFLANPPPQEFWTLDKMLTSPSPEQSGFSSLYKLLLEVLPQKCGSTEGEFIYTVERMLKDCPSSKQAMALLAKIKVPSSKAPSVTLNECFPTDVPVNSELISWEEPKDPGAAVVLCPSDAKESKETAPEEAQARNRKVLHPVVLTEELSEQQVQVVEGDQDMPLDLTWPTLTATATPVRGPVPDNLSSGIGAQPATVQELILARPAPRLVERCGTESNGSSSFLDLPDVQTSDQPLDLSLAAWPVRATASDPGWIHGRREPVFVKPRGVFSDGESALQFGELSEASSVVDDRTKEAPVVDAPIDLTTSNETLSGSDPFEFAKFRRPRFSAQALIDRGGPLADVHAKIKSRVYEQCLQACEPGSRATPATKKWLDKMWDRVDMKTWRCTSQFQAGHILESLKFLPDMIQDTPPPVPRKNRAGDSAGLKQLVAQWDRKLSVTPPTKPVGPVLDQTVPLPMDIQQEDAISADKPPHSQNPSSQVDVGGGWKSFMLSGTRFAGSVSQRLTTWVFEVLSHLPAFMLTLFSPRGSMAPGDWLFAGAVLLALLLCRSYPILGCLPLLGVFSGSVRCVRLGVFGSWMAFAVFLFSTPPDPVGSSCDHDSPECHAELLALEQRQLWEPVRSLVVGPSGLLCVILGKLLGGSRCLWFVLLRICMLADLAISLIYVVSQGRCHKCWGKCIRTAPAEVTLNVFPFSRATRSSLVSLCDRFQAPKGVDPVHLATGWRGCWCGESPIHQSHQKPIAYANLDEKKISAQTVIAVPYDPSQAIKCLKVLQAGGAIVDQPTPEVVRVSEIPFSAPFFPKVPVNPDCRVVVDSDTFVAAVRCGYSTAQLVLGRGNFAKLNQTPLRNSVPTKTTGGASYTLAVAQVSVWTLVHFILGLWLTSPQVCGRGTSDPWCSNPFSYPTYGPGVVCSSRLCVSADGVTLPLFSAVAHLSGREVGIFILVLASLGALAHRLALKADMSMVFLAFCAYAWPMSSWLICFFPMLLRWVTLHPLTMLWVHSFLVFCLPAAGVLSLGITGLLWAVGRFTQVAGIITPYDIHQYTSGPRGAAAVATAPEGTYMAAVRRAALTGRTLIFTPSAVGSLLEGAFRTQKPCLNTVNVVGSSLGSGGVFTIDGRRVIVTATHVLNGNTARVTGDSYNRMHTFNTNGDYAWSHADDWQGVAPMVKIAKGYRGRAYWQTSTGVEPGIMGEGFAFCFTNCGDSGSPVISEAGDLIGVHTGSNKLGSGLVTTPEGETCSIKETRLSDLSRHFAGPSVPLGDIKLSPAIIPDVTTIPSDLASLLASVPVMEGGLSTVQLLCVFFLLWRMMGHAWTPIVAVGFFLLNEILPAVLVRAVFSFALFVLAWATPWSAQVLMIRLLTAALNRNRLSLAFYALGGVVGLATEIGTFAGGWPELSQALSTYCFLPRFLAVTSYVPTIIIGGLHALGVILWLFKYRCLHNMLVGDGSFSSAFFLRYFAEGNLRKGVSQSCGMNNESLTAALACKLSQADLDFLSSLTNFKCFVSASNMKNAAGQYIEAAYARALRQELASLVQVDKMKGVLAKLEAFAETATPSLDTGDVIVLLGQHPHGSILDINVGGERKTVSVQETRCLGGSKFSVCTVVSNTPVDTLTGIPLQTPTPLFENGPRHRSEDDDLKVERMKKHCVSLGFHKINGKVYCKIWDKSNGDTFYTDDSRYTQDHAFQDRSTDYRDRDYEGVQTAPQQGFDPKSEAPVGTVVIGGITYNRHLVKGKEVLVPKPDNCLEAARLSLEQALAGMGQTCDLTATEVEKLKRIISQLQGLTTEQALNCSEQ ID NO: 12 ORF 1B OF PARENTAL PRRSV STRAIN 94881 ENCODED BYSEQUENCE OF SEQ ID NO: 10 BETWEEN NUCLEOTIDES 7209 . . . 11600TGFKLLAASGLTRCGRGGLVVTETAVKIVKYHSRTFTLGSLDLKVTSEVEVKKSTEQGHAVVANLCSGVVLMRPHPPSLVDVLLKPGLDTTPGIQPGHGAGNMGVNGSIWDFETAPTKVELELSKQIIQACEVRRGDAPNLQLPYKLYPVRGDPERRKGRLVNTRFGDLPYKTPQDTKSAIHAACCLHPNGVLVSDGKSTLGTTLQHGFELYVPTVPYSVMEYLDSRPDTPFMCTKHGTSKAAAEDLQKYDLSTQGFVLPGVLRLVRRFIFSHVGKAPPLFLPSTYPAKNSMAGVNGQRFPTKDVQSIPEIDEMCARAVKENWQTVTPCTLKKQYCSKPKTRTILGTNNFIALAHRSALSGVTQAFMKKAWKSPIALGKNKFKELHCTVAGRCLEADLASCDRSTPAIVRWFVANLLYELAGCEEYLPSYVLNCCHDLVATQDGAFTKRGGLSSGDPVTSVSNTVYSLIIYAQHMVLSALKMGHEIGLKFLEEQLKFEDLLEIQPMLVYSDDLVLYAERPTFPNYHWWVEHLDLMLGFKTDPKKTVITDKPSFLGCRIEAGRQLVPNRDRILAALAYHMKAQNASEYYASAAAILMDSCACIDHDPEWYEDLICGIARCARQDGYRFPGPAFFMSMWEKLKSHNEGKKCRHCGICDAKADYASACGLDLCLFHSHFHQHCPVTLSCGHHAGSKECSQCQSPVGAGKSPLDAVLKQIPYKPPRTIIMKVDNKTTTLDPGRYQSRRGLVAVKRGIAGNEVDLSDGDYQVVPLLPTCKDINMVKVACNVLLSKFIVGPPGSGKTTWLLNQVQDDDVIYTPTHQTMFDIVSALKVCRYSIPGASGLPFPPPARSGPWVRLIASGHVPGRVSYLDEAGYCNHLDILRLLSKTPLVCLGDLQQLHPVGFDSYCYVFDQMPQKQLTTIYRFGPNICAAIQPCYREKLESKARNTRVVFTTRPVAFGQVLTPYHKDRTGSAITIDSSQGATFDIVTLHLPSPKSLNKSRALVAITRARHGLFIYDPHDQLQEFFNLTPERTDCNLAFSRGDELVVLNVDNAVTTVAKALETGSPRFRVSDPRCKSLLAACSASLEGSCMPLPQVAHNLGFYFSPDSPAFAPLPKELAPHWPVVTHQNNRAWPDRLVASMRPIDARYSKPMVGAGYVVGPSIFLGTPGVVSYYLTLYIGGEPQALPETLVSTGRIATDCREYLDAAEEEAARELPHAFIGDVKGTTVGGCHHITSKYLPRSLPKDSVAVVGVSSPGRAAKAVCTLTDVYLPELRPYLQPETASKCWKLKLDFRDVRLMVWKGATAYFQLEGLTWSALPDYARFIQLPKDAVVYIDPCIGPATANRKVVRTTDWRADLAVTPYDYGAQVILTTAWFEDLGPQWKILGLQPFRRTFGFENTEDWAILARRMNDGKDYTDYNWHCVRERPHAIYGRARDHTYHFALGTELQVELGRPRLPPEQVPSEQ ID NO: 13 ORF 2 OF PARENTAL PRRSV STRAIN 94881 ENCODED BYSEQUENCE OF SEQ ID NO: 10 BETWEEN NUCLEOTIDES 11611 . . . 12360MQWVHCGVKSVSCSWMPSLSSLLVWLTLSSFSPYCLGSLLQAGYWSSFSEWFAPRFSVRALPFTLPNYRRSYEGLLPNCRPDVPQFAVKHPLGILWHMRVSHLIDEMVSRRIYRTMEHSGQAAWKQVVSEATLTKLSRLDVVTHFQHLAAVEADSCRFLSSRLAMLKNLAVGNVSLEYNTTLDRVELIFPTPGTRPKLTDFRQWLISVHASIFSSVASSVTLFTVLWLRIPALRYVFGFHWPTATHHSNSEQ ID NO: 14 ORF 3 OF PARENTAL PRRSV STRAIN 94881 ENCODED BYSEQUENCE OF SEQ ID NO: 10 BETWEEN NUCLEOTIDES 12219 . . . 13016MAYQRARFHLLLCGFVCYLVHSALASNSSSTLCFWFPLAHGNTSFELTINYTICKPCPTSQAAQQRLEPGRNVWCKIGHDRCEERDHDELSMSIPSGYDNLKLEGYYAWLAFLSFSYAAQFHPELFGIGNVSRVFVDKRHQFICAEHDGQNSTISARHNISASYAVYYHHQIDGGNWFHLEWLRPFFSSWLVLNISWFLRRSPASPASRRIYQILRPTRPRLPVSWSFRTSIVSNLTGPQQRKVPLPSGGRPNVVKPSAFPSTSRSEQ ID NO: 15 ORF 4 OF PARENTAL PRRSV STRAIN 94881 ENCODED BYSEQUENCE OF SEQ ID NO: 10 BETWEEN NUCLEOTIDES 12761 . . . 13312MAATILFLLAGAQHLMVSEAFACKPCFSTHLSDIKTNTTAAAGFMVLQNINCFQSHRASTAQGTTPLRRSSQCREAVGIPQYITITANVTDESYLYNADLLMLSACLFYASEMSEKGFKVIFGNISGVVSACVNFTDYVAHVTQHTQQHHLVIDHIRLLHFLTPSTMRWATTIACLFAILLAVSEQ ID NO: 16 ORF 5 OF PARENTAL PRRSV STRAIN 94881 ENCODED BYSEQUENCE OF SEQ ID NO: 10 BETWEEN NUCLEOTIDES 13309 . . . 13914MKCSCKLGHFLTPHSCFWWLFLLCTGLSWSFVDGNDNSSTSQYIYNLTICELNGTEWLSGHFDWAVETFVLYPVATHIISLGFLTTSHFLDALGLGAVSATGFIGERYVLSSMYGVCAFAALVCFVIRAAKNCMACRYARTRFTNFIVDDRGRIHRWKSSIVVEKLGKAEVGGDLVNIKHVVLEGVKAQPLTRTSAEQWEASEQ ID NO: 17 ORF 6 OF PARENTAL PRRSV STRAIN 94881 ENCODED BYSEQUENCE OF SEQ ID NO: 10 BETWEEN NUCLEOTIDES 13902 . . . 14423MGSLDDFCNDPTAAQKLVLAFSITYTPIMIYALKVSRGRLLGLLHILIFLNCSFTFGYMTYVHFQSTNRVALTLGAVVALLWGVYSLTESWKFITSRCRLCCLGRRYILAPAHHVESAAGLHSIPASGNRAYAVRKPGLTSVNGTLVPGLRSLVLGGKRAVKRGVVNLVKYGRSEQ ID NO: 18 ORF 7 OF PARENTAL PRRSV STRAIN 94881 ENCODED BYSEQUENCE OF SEQ ID NO: 10 BETWEEN NUCLEOTIDES 14413 . . . 14799MAGKNQSQKKRRNAAPMGKGQPVNQLCQLLGTMIKSQRQQSRGGQAKKKKPEKPHFPLAAEDDIRHHLTQAERSLCLQSIQTAFNQGAGTASLSSSGKVSFQVEFMLPVAHTVRLIRVTSTSASQGANSEQ ID NO: 19 Nucleotide encoding attenuated PRRSV 94881 ORF1A 178 ATG181 TCTGGGATGT TCTCCCGGTG CATGTGCACC CCGGCTGCCC GGGTATTTTG GAACGCCGGC241 CAAGTCTATT GCACACGGTG TCTCAGTGCA CGGTCTCTTC TCTCTCCAGA ACTTCAGGAC301 ACGGACCTCG GTGCAGTTGG CTTGTTTCAC AAGCCTAAAG ACAAGCTCCA TTGGAAAGTT361 CCCATTGGTA TCCCCCAGGT GGAATGTTCT CCATCTGGGT GTTGCTGGCT GTCAACCATT421 TTTCCTTTAG CGCGCATGAC CTCCGGCAAT CACAACTTCC TTCAACGACT CGTGAAGGTT481 GCTGATGTAT TGTACCGTGA CGGTTGCTTA ACCCCTAGAC ACCTCCGTGA ACTCCAAGTT541 TACGAGCGTG GTTGCAATTG GTATCCGATT ACGGGGCCTG TGCCTGGGAT GGCTGTGTAC601 GCGAACTCCA TGCACGTGTC CGACCAACCG TTCCCTGGTG CCACTCATGT GTTAACAAAT661 TCCCCTTTGC CTCAACGGGC TTGTCGGCAG CCGTTCTGTC CGTTCGAAGA GGCCCATTCT721 AGCATATACA GGTGGGAAAA ATTTGTAATT TTTATGGATT CCTCCTCCGA CGGTCGATCT781 CGCATGATGT GGACTCCGGA ATCCGATGAC TCCACGGCTT TGGAAGTTCT GCCGCCCGAG841 CTAGAACACC AGGTCAAGGT CCTTGTTCGG AGCTTTCCCG CCCATCACCT TGTCGACCTT901 GCCGATTGGG AGCTCACTGA GTCCCCTGAT AACGGTTTTT CCTTCAGCAC GTCACATCCT961 TGCGGCTACC TTGTTCGGGA CCCGGCTGTA TCCGAAGGCA AGTGTTGGCT TTCCTGCTTT1021 TTGAGCCAGT CAGCCGAAGT GCTCAGTCGC GAGGCGCATC TGGCTACCGC CTATGGTTAC1081 CAAACCAAGT GGGGTGTGCC TGGCAAGTAC ATCCAGCGCA GACTTCAAGT TCACGGTCTC1141 CGTGCTGTGG TCGACCCTGA TGGTCCCATT CACGTTGAAG CATTGTCTTG CCCCCAGTCT1201 TGGATCAGGC ACTTGACCCT GAATGATGAT GTCACCCCGG GATTCGTTCG CCTAATGTCT1261 CTTCGCATTG TGCCGAACAC AGAGCCTACC ACACACCGGA TCTTTCGTTT TGGAGTGCAC1321 AAGTGGTATG GTGCCGCCGG CAAACGGGCC CGTGGCAAGC GTGCCGCCAA AAGTGAGAAA1381 GACTCGGCTT CCACCCTCAA GGTTGCCCGA CCGACTTCCA CCAGTGGAAT CGTCACCTAC1441 TCCCCACCTG CGGACGGGTC TTGTGGTTGG CATGCCCTTG CCGCCATACT GAACCGGATG1501 ATTAATAATG ACTTCACGTC CCCTCTGCCT CGGTACAACA GGCCGGAGGA CGATTGGGCT1561 TCTGATGGTG ACCTTGCTCA GGCCATTCAA TGTTTGCAAC TACCTGCCGC CATAGCTCGG1621 AACCGCGCCT GCCCTAACGC CAAATACCTC ATAAAACTCA ACGGAGTTCA TTGGGAGGTA1681 GAGGTGAGGC CTGGAATGGC TCCTCGCTCC CTCTCTCGTG AGTGCGTTGT TGGCGTCTGC1741 TCTGAAGGCT GTGTCGCGTC GCCTTACCCG GAGGACGGGT TGCCTAAACG TGCACTTGAG1801 GCCCTGGCGT CTGCTTATAG ACTGCCTTCA GACTGTGTTT GTGATGGTAT TATTGACTTC1861 CTTGCCAATC CACCTCCCCA GGAGTTCTGG ACTCTTGACA AAATGTTGAC TTCCCCGTCA1921 CCGGAGCAGT CCGGCTTCTC TAGTCTGTAT AAATTGTTGT TAGAGATCTT GCCGCAGAAA1981 TGCGGATCCA CAGAAGGGGA ATTCATCTAT ACTGTTGAGA GGATGTTGAA GGATTGTCCG2041 AGCTCCAAAC AGGCCATGGC CCTCCTTGCA AAAATTAAGG TCCCATCCTC AAAGGCCCCA2101 TCCGTGACTC TGAACGAGTG CTTCCCCACG GATGTTCCAG TCAACTCTGA GTTAATATCT2161 TGGGAAGAGC CCAAAGACCC TGGCGCTGCT GTTGTCCTAT GTCCATCGGA TGCAAAAGAA2221 TCTAAGGAAA CAGCCCCTGA AGAAGCTCAA GCGAGAAACC GTAAGGTCCT TCACCCTGTG2281 GTCCTTACCG AGGAACTTAG CGAGCAACAG GTGCAGGTGG TTGAGGGTGA TCAGGATATG2341 CCACTGGATT TGACTTGGCC AACCTTAACC GCTACGGCGA CCCCTGTTAG AGGGCCGGTA2401 CCGGACAATT TGAGCTCTGG CATTGGTGCC CAGCCCGCTA CCGTTCAAGA ACTCATTCTG2461 GCGAGGCCTG CACCCCGTCT TGTTGAGCGC TGTGGCACGG AGTCGAACGG CAGCAGTTCA2521 TTTCTGGATT TGCCTGACGT GCAGACCTCG GACCAGCCTT TAGACCTGTC CCTGGCCGCG2581 TGGCCTGTAA GGGCTACCGC GTCTGACCCC GGTTGGATCC ACGGTAGGCG TGAGCCTGTC2641 TTTGTGAAGC CTCGAGGTGT TTTCTCTGAT GGCGAGTCGG CCCTTCAGTT CGGAGAGCTT2701 TCCGAAGCCA GTTCTGTCGT CGATGACCGG ACAAAAGAAG CTCCGGTGGT TGACGCCCCC2761 ATCGATTTGA CAACTTCGAA CGAGACGCTC TCTGGGTCTG ACCCCTTTGA ATTCGCCAAA2821 TTCAGGCGCC CGCGTTTCTC CGCGCAAGCT TTAATCGACC GAGGTGGTCC GCTTGCCGAT2881 GTTCATGCAA AGATAAAGAG TCGGGTATAT GAACAATGCC TTCAAGCTTG TGAACCTGGT2941 AGTCGTGCGA CCCCAGCCAC CAAGAAGTGG CTCGACAAAA TGTGGGACAG GGTGGACATG3001 AAAACTTGGC GCTGCACCTC GCAGTTCCAA GCTGGTCACA TTCTTGAGTC CCTCAAATTC3061 CTCCCTGACA TGATTCAAGA CACACCGCCT CCTGTTCCCA GGAAGAACCG AGCTGGTGAC3121 AGTGCCGGCC TGAAGCAACT GGTGGCGCAG TGGGATAGGA AATCGAGTGT GACACCCCCC3181 ACAAAACCGG TTGGACCGGT GCTTGACCAG GCCGTCCCTC TGCCTATGGA CATCCAGCAA3241 GGAGATGCCA TCTCCGCTGA CAAGCCACCC CATTCGCAAA ACCCTTCTAG TCAAGTAGAT3301 GTGGGTGGAG GTTGGAAAAG TTTTATGCTC TCCGGCACCC GTTTCGCGGG GTCCGTTAGT3361 CAGCGCCTTA CGACATGGGT TTTTGAGGTT CTCTCCCATC TCCCAGCTTT TATGCTCACA3421 CTTTTCTCGC CACGGGGCTC TATGGCTCCA GGTGATTGGC TGTTTGCAGG TGCTGTTCTA3481 CTTGCTCTCC TGCTCTGCCG TTCTTACCCA ATACTCGGAT GCCTTCCCTT ATTGGGTGTC3541 TTTTCTGGTT CTGTGCGGTG TGTTCGTTTG GGTGTTTTTG GTTCTTGGAT GGCTTTTGCT3601 GTATTTTTAT TCTCGACTCC ACCCGACCCA GTCGGTTCTT CTTGTGACCA CGATTCGCCG3661 GAGTGTCATG CTGAGCTTTT GGCTCTTGAG CAGCGCCAAC TTTGGGAACC TGTGCGCAGC3721 CTTGTGGTCG GGCCATCGGG CCTCTTATGC GTCATTCTTG GCAAGTTACT CGGTGGGTCA3781 CGTTGTCTCT GGTTTGTTCT CCTACGTATA TGCATGCTCG CAGATTTGGC AATTTCTCTT3841 ATTTATGTGG TGTCCCAAGG GCGTTGTCAC AAGTGTTGGG GAAAGTGTAT AAGGACGGCT3901 CCTGCAGAAG TGGCCCTTAA TGTGTTTCCT TTTTCGCGCG CCACCCGCTC ATCTCTTGTG3961 TCCTTGTGTG ATCGGTTCCA AGCGCCAAAA GGAGTTGACC CCGTGCACTT GGCGACAGGC4021 TGGCGCGGGT GCTGGTGTGG TGAGAGCCCT ATTCATCAAT CACACCAAAA ACCGATAGCT4081 TATGCCAACT TGGATGAAAA GAAGATATCC GCCCAGACGG TGATTGCTGT CCCGTATGAT4141 CCTAGTCAGG CCATTAAATG CCTGAAAGTT TTGCAGGCAG GAGGGGCTAT TGTGGACCAG4201 CCTACGCCCG AGGTCGTCCG TGTGTCTGAG ATTCCCTTCT CGGCCCCATT TTTTCCGAAG4261 GTCCCAGTCA ACCCAGACTG CAGGGTTGTG GTAGATTCGG ACACTTTTGT GGCTGCGGTC4321 CGCTGCGGTT ATTCGACAGC ACAACTGGTC CTTGGTCGGG GCAACTTTGC CAAGCTAAAT4381 CAGACCCCCC TCAGGAACTC TGTCCCCACC AAAACAACTG GTGGGGCCTC ATACACCCTT4441 GCCGTGGCCC AGGTATCTGT GTGGACTCTT GTTCATTTCA TCCTCGGCCT TTGGTTAACG4501 TCACCTCAAG TGTGTGGTCG AGGGACCTCT GACCCGTGGT GTTCGAACCC TTTTTCGTAT4561 CCTACTTATG GCCCCGGAGT TGTGTGTTCC TCTCGACTCT GCGTGTCTGC CGACGGAGTT4621 ACCCTGCCAT TGTTCTCAGC CGTTGCCCAT CTTTCCGGTA GAGAGGTGGG GATTTTTATT4681 TTGGTGCTTG CCTCCTTGGG CGCTTTAGCC CACCGCTTGG CTCTTAAGGC AGACATGTCA4741 ATGGTCTTTT TGGCGTTTTG TGCTTACGCC TGGCCCATGA GCTCCTGGTT AATTTGCTTC4801 TTTCCTATGC TCTTGAGGTG GGTAACCCTT CATCCTCTCA CTATGCTTTG GGTGCACTCA4861 TTTTTGGTGT TTTGCCTACC AGCTGCCGGC GTTCTCTCGC TGGGAATAAC CGGTCTTCTT4921 TGGGCAGTTG GCCGTTTCAC CCAGGTTGCC GGAATTATCA CACCTTATGA CATCCACCAG4981 TATACCTCCG GACCACGTGG TGCAGCTGCT GTAGCAACGG CTCCAGAAGG TACTTACATG5041 GCGGCCGTTC GGAGAGCCGC TTTGACTGGA CGGACTTTGA TCTTCACACC ATCTGCAGTC5101 GGATCCCTTC TTGAAGGTGC TTTCAGAACT CAAAAGCCCT GCCTTAACAC CGTGAATGTC5161 GTAGGCTCTT CCCTTGGTTC TGGAGGAGTT TTCACCATTG ATGGCAGAAG AGTCATCGTC5221 ACTGCCACCC ATGTGTTGAA TGGTAACACA GCCAGGGTCA CTGGTGATTC CTACAACCGC5281 ATGCACACGT TCAATACTAA TGGTGATTAT GCCTGGTCCC ATGCTGATGA CTGGCAAGGC5341 GTTGCCCCTA TGGTTAAGAT CGCTAAGGGG TATCGCGGTC GTGCCTACTG GCAAACGTCA5401 ACCGGAGTCG AACCTGGCAT CATGGGGGAA GGATTCGCCT TCTGTTTCAC TAACTGTGGC5461 GACTCAGGGT CACCTGTCAT TTCAGAAGCT GGTGACCTTA TTGGAGTCCA TACCGGTTCA5521 AACAAACTCG GTTCTGGTCT TGTGACAACC CCTGAAGGGG AGACCTGCTC CATCAAGGAA5581 ACTAGGCTCT CTGACCTTTC TAGACATTTT GCAGGTCCAA GCGTCCCTCT TGGGGACATT5641 AAGTTGAGCC CAGCCATCAT CCCTGATGTG ACAACTATTC CGAGTGACTT GGCATCGCTC5701 CTTGCTTCTG TCCCCGTGAT GGAAGGTGGC CTCTCAACTG TCCAGCTTTT GTGCGTCTTT5761 TTCCTTCTCT GGCGCATGAT GGGCCATGCC TGGACACCCA TTGTTGCCGT AGGCTTCTTT5821 TTGCTGAATG AAATTCTCCC AGCAGTCTTG GTCCGAGCTG TGTTCTCTTT TGCACTCTTT5881 GTACTTGCAT GGGCCACCCC CTGGTCGGCA CAAGTGTTGA TGATTAGACT CCTCACGGCG5941 GCTCTCAACC GCAACAGGTT GTCCCTGGCG TTCTACGCAT TCGGAGGTGT CGTTGGCCTG6001 GCCACAGAAA TCGGGACTTT TGCTGGTGGA TGGCCTGAAC TGTCCCAAGC CCTCTCGACA6061 TACTGCTTCC TGCCCAGGTT CCTTGCTGTG ACTAGTTATG TCCCCACCAT CATCATCGGT6121 GGGCTCCATG CCCTCGGCGT AATTTTGTGG TTATTCAAAT ACCGATGCCT CCACAACATG6181 CTGGTTGGTG ATGGGAGTTT CTCAAGCGCT TTCTTCCTAC GGTATTTTGC TGAGGGTAAT6241 CTTAGGAAAG GCGTGTCGCA GTCCTGTGGC ATGAATAACG AATCCCTGAC AGCTGCTTTG6301 GCTTGCAAGT TGTCGCAAGC TGACCTTGAT TTTTTGTCCA GTTTAACGAA CTTCAAGTGC6361 TTTGTGTCCG CTTCAAACAT GAAAAATGCA GCTGGCCAAT ACATCGAGGC GGCGTATGCT6421 AGAGCTCTGC GTCAGGAGCT GGCCTCCTTG GTTCAGGTTG ACAAGATGAA AGGAGTATTG6481 GCCAAGCTCG AGGCTTTCGC TGAGACGGCC ACTCCGTCAC TTGACACAGG GGACGTGATT6541 GTTCTGCTTG GGCAACACCC CCATGGATCC ATCCTCGACA TTAATGTGGG GGGTGAAAGG6601 AAAACTGTGT CTGTGCAAGA AACACGATGC CTGGGTGGTT CCAAATTCAG TGTCTGCACT6661 GTCGTGTCCA ACACGCCCGT GGATACCTTG ACCGGTATCC CACTTCAGAC GCCAACCCCA6721 CTTTTTGAAA ATGGCCCGCG CCATCGCAGC GAGGACGACG ACCTCAAAGT TGAGAGAATG6781 AAAAAACACT GTGTATCCCT CGGCTTCCAC AAAATCAATG GTAAAGTTTA CTGCAAAATT6841 TGGGACAAGT CTAACGGCGA CACCTTTTAC ACGGATGATT CCCGATACAC TCAAGACCAT6901 GCTTTTCAGG ACAGGTCAAC CGACTATAGA GACAGGGATT ATGAAGGTGT ACAGACCGCC6961 CCCCAACAGG GATTCGATCC AAAGTCCGAA GCCCCTGTTG GCACTGTTGT AATCGGTGGC7021 ATTACGTATA ACAGGCATCT GGTCAAAGGT AAGGAGGTCC TAGTTCCCAA ACCTGACAAC7081 TGCCTTGAAG CTGCCAGACT GTCCCTTGAG CAAGCTCTTG CTGGGATGGG CCAAACTTGT7141 GACCTTACAG CTACCGAAGT GGAGAAACTA AAGCGCATCA TTAGTCAACT CCAAGGTCTG7201 ACCACTGAAC AGGCTTTAAA CTGCTAGSEQ ID NO: 20 Nucleotide encoding attenuated PRRSV 94881 ORF1B 7209AC AGGCTTTAAA CTGCTAGCCG CCAGCGGCTT GACCCGCTGT GGCCGCGGCG 7261GCCTAGTTGT AACTGAAACG GCGGTAAAAA TCGTAAAATA CCACAGCAGA ACTTTCACCT 7321TAGGCTCTTT AGACCTAAAA GTCACCTCCG AGGTGGAGGT GAAGAAATCA ACTGAGCAGG 7381GGCACGCTGT CGTGGCGAAC TTATGTTCCG GTGTCGTCTT GATGAGGCCT CACCCACCGT 7441CCCTTGTTGA CGTTCTCCTC AAACCCGGAC TTGACACAAC ACCCGGCATT CAACCAGGGC 7501ATGGGGCCGG GAATATGGGC GTGAACGGTT CTATTTGGGA TTTTGAAACT GCACCCACAA 7561AGGTAGAACT AGAGTTGTCC AAGCAAATAA TCCAAGCATG TGAAGTCAGG CGCGGGGACG 7621CCCCTAACCT CCAACTCCCC TACAAGCTTT ATCCTGTCAG GGGGGACCCC GAGCGGCGTA 7681AAGGTCGCCT TGTCAACACT AGGTTTGGAG ATTTACCTTA CAAAACTCCC CAAGACACCA 7741AGTCCGCAAT TCATGCGGCT TGTTGCCTGC ATCCCAATGG GGTCCTCGTG TCTGATGGCA 7801AATCCACGCT GGGTACCACT CTTCAACATG GTTTCGAGCT TTATGTCCCC ACTGTACCTT 7861ATAGTGTCAT GGAATACCTT GATTCACGCC CTGACACCCC TTTTATGTGT ACTAAACATG 7921GCACTTCCAA GGCTGCTGCA GAGGACCTCC AAAAATATGA CCTATCCACT CAAGGGTTTG 7981TCTTGCCTGG GGTCCTACGC CTAGTGCGCA GGTTCATCTT TAGCCATGTT GGTAAGGCGC 8041CACCACTGTT CCTTCCATCA ACCTACCCTG CCAAGAACTC CATGGCAGGG GTCAATGGCC 8101AGAGGTTCCC AACAAAGGAT GTCCAGAGCA TACCTGAAAT TGATGAAATG TGCGCCCGTG 8161CCGTCAAGGA AAATTGGCAG ACTGTGACAC CTTGCACCCT CAAAAAACAG TACTGTTCCA 8221AACCTAAAAC TAGAACCATC CTAGGTACCA ACAACTTCAT AGCCTTGGCT CACAGGTCAG 8281CACTCAGTGG TGTCACCCAG GCGTTCATGA AGAAGGCCTG GAAGTCCCCA ATTGCCTTGG 8341GGAAAAACAA GTTTAAGGAA TTGCATTGCA CTGTCGCCGG CAGATGCCTT GAGGCTGACC 8401TGGCTTCCTG CGATCGCAGC ACCCCCGCCA TTGTGAGGTG GTTTGTTGCC AACCTCCTGT 8461ATGAACTTGC AGGATGTGAA GAGTACTTGC CTAGCTACGT GCTCAACTGT TGCCATGACC 8521TTGTGGCAAC GCAGGATGGC GCTTTCACAA AACGCGGTGG CCTGTCGTCC GGGGACCCCG 8581TCACCAGTGT GTCCAACACC GTCTACTCAC TGATAATTTA CGCCCAGCAC ATGGTGCTTT 8641CGGCCTTGAA GATGGGTCAT GAAATTGGTC TCAAGTTCCT TGAGGAACAG CTCAAATTTG 8701AGGACCTTCT TGAAATCCAG CCCATGTTAG TGTATTCTGA TGACCTCGTC TTGTATGCGG 8761AAAGACCCAC TTTTCCCAAC TACCATTGGT GGGTCGAGCA TCTTGACCTG ATGTTGGGCT 8821TTAAAACGGA CCCAAAGAAA ACTGTCATAA CTGATAAACC CAGTTTTCTC GGCTGCAGAA 8881TTGAAGCAGG ACGGCAGTTA GTCCCCAATC GCGACCGTAT TCTGGCTGCT CTTGCATATC 8941ATATGAAGGC GCAGAACGCC TCAGAGTATT ATGCGTCCGC TGCCGCAATT CTGATGGATT 9001CGTGTGCTTG CATTGACCAT GACCCCGAGT GGTATGAGGA TCTTATCTGC GGCATCGCCC 9061GGTGTGCTCG CCAGGACGGT TACCGTTTTC CAGGCCCGGC ATTTTTCATG TCCATGTGGG 9121AGAAGCTGAA AAGTCATAAT GAAGGGAAGA AATGCCGTCA CTGCGGCATC TGCGACGCCA 9181AAGCCGACTA TGCGTCCGCC TGTGGACTTG ATTTGTGTTT GTTCCATTCA CACTTTCATC 9241AACACTGCCC AGTCACTCTG AGCTGTGGCC ACCATGCCGG TTCAAAGGAA TGTTCGCAGT 9301GTCAGTCACC TGTCGGGGCT GGCAAATCCC CCCTTGACGC TGTGCTGAAA CAAATCCCGT 9361ACAAACCTCC TCGTACCATT ATCATGAAGG TGGACAACAA AACAACGACC CTTGACCCGG 9421GAAGATATCA GTCCCGTCGA GGTCTTGTTG CAGTCAAAAG AGGTATTGCA GGTAATGAGG 9481TTGATCTTTC TGATGGAGAC TACCAAGTGG TGCCTCTTTT GCCGACTTGC AAAGACATAA 9541ACATGGTGAA GGTGGCTTGC AACGTACTAC TCAGCAAGTT TATAGTAGGG CCGCCAGGTT 9601CCGGAAAAAC CACCTGGCTA CTGAACCAAG TCCAGGACGA TGATGTCATT TACACACCTA 9661CTCATCAGAC AATGTTTGAC ATAGTCAGTG CTCTTAAAGT TTGCAGGTAT TCCATCCCAG 9721GAGCCTCAGG ACTCCCTTTT CCACCACCTG CCAGGTCCGG GCCGTGGGTT AGGCTCATCG 9781CCAGCGGACA TGTCCCTGGC CGAGTGTCAT ATCTCGATGA GGCAGGATAT TGCAATCATC 9841TAGACATTCT AAGGCTGCTT TCCAAAACAC CCCTTGTGTG TTTGGGTGAC CTTCAGCAAC 9901TTCACCCGGT CGGCTTTGAT TCCTATTGTT ATGTGTTCGA TCAGATGCCT CAGAAGCAGC 9961TGACCACCAT TTATAGATTT GGCCCTAACA TCTGTGCAGC CATCCAGCCT TGTTACAGGG 10021AGAAACTTGA ATCCAAGGCC AGGAACACCA GAGTGGTTTT CACCACCCGG CCTGTGGCCT 10081TTGGTCAGGT CCTGACACCG TACCACAAAG ATCGTACCGG CTCTGCAATA ACTATAGATT 10141CATCCCAGGG GGCGACCTTC GACATTGTGA CATTGCATCT ACCATCGCCA AAGTCCCTAA 10201ACAAATCCCG AGCACTTGTA GCCATCACTC GGGCAAGACA TGGGTTGTTC ATTTATGACC 10261CTCATGACCA ACTCCAGGAG TTTTTCAACT TAACCCCCGA GCGCACTGAT TGTAACCTTG 10321CGTTCAGCCG TGGGGATGAG CTGGTTGTTT TGAATGTGGA TAATGCGGTC ACAACTGTAG 10381CGAAGGCCCT AGAGACAGGT TCACCCCGAT TTCGAGTATC GGACCCGAGG TGCAAGTCTC 10441TCTTAGCCGC TTGTTCGGCC AGTCTAGAAG GGAGCTGCAT GCCACTACCA CAAGTAGCAC 10501ATAACCTGGG GTTTTACTTT TCCCCGGACA GCCCAGCTTT TGCACCCCTG CCAAAAGAGC 10561TGGCGCCACA TTGGCCAGTG GTCACCCACC AGAATAATCG AGCGTGGCCT GATCGACTTG 10621TCGCTAGTAT GCGCCCAATT GATGCCCGCT ACAGCAAGCC AATGGTCGGT GCAGGGTATG 10681TGGTCGGGCC ATCCATTTTT CTTGGCACTC CTGGTGTGGT GTCATACTAT CTCACATTAT 10741ACATCGGGGG CGAGCCTCAG GCCCTGCCAG AAACACTCGT TTCAACAGGA CGTATAGCCA 10801CAGATTGTCG GGAATATCTC GACGCGGCTG AGGAAGAGGC AGCGAGAGAA CTTCCCCACG 10861CATTTATTGG CGATGTCAAA GGCACTACGA TCGGGGGGTG TCACCACATT ACATCGAAAT 10921ACCTACCTAG GTCCCTGCCT AAAGACTCTG TTGCTGTGGT TGGGGTGAGT TCGCCCGGTA 10981GGGCTGCTAA AGCCGTGTGC ACTCTCACCG ATGTGTACCT CCCCGAACTC CGACCATATT 11041TGCAACCGGA GACGGCATCA AAATGCTGGA AACTTAAACT GGATTTCAGG GATGTTCGAC 11101TGATGGTCTG GAAAGGCGCC ACAGCCTATT TCCAGTTGGA AGGGCTGACA TGGTCAGCGC 11161TGCCCGATTA TGCTAGGTTC ATTCAGCTAC CCAAGGATGC CGTTGTGTAC ATCGATCCGT 11221GTATAGGGCC GGCAACAGCC AATCGCAAGG TTGTGCGAAC CACAGACTGG CGGGCCGACC 11281TGGCAGTGAC ACCGTATGAT TACGGTGCTC AGGTCATTTT GACAACAGCC TGGTTCGAGG 11341ACCTTGGGCC GCAGTGGAAG ATTTTGGGGT TGCAGCCTTT CAGACGAACA TTTGGCTTTG 11401AGAACACTGA AGATTGGGCA ATTCTCGCAC GCCGTATGAA TGACGGCAAA GATTACACTG 11461ACTATAATTG GCATTGTGTA CGAGAACGCC CACACGCAAT TTACGGGCGC GCCCGTGACC 11521ATACGTATCA TTTTGCCCTT GGCACTGAAC TGCAAGTAGA GCTGGGCAGA CCCCGGCTGC 11581CTCCTGAGCA AGTGCCGTGASEQ ID NO: 21 Nucleotide encoding attenuated PRRSV 94881 ORF2 11611ATGCAATGGG TTTACTGTGG AGTAAAATCA 11641GTCAGTTGTT CGTGGATGCC TTCACTGAGT TCCTTGTTAG TGTGGTTGAC ATTGTCATCT 11701TTCTCGCCAT ATTGTTTGGG TTCACTGTTG CAGGCTGGTT ATTGGTCTTC CTTCTCAGAG 11761TGGTTTGCTC CGCGTTTCTC CGTTCGCGCT CTGCCATTCA CTCTTCCGAA CTATCGAAGG 11821TCCTATGAGG GCTTGCTACC CAACTGCAGA CCGGATGTCC CACAATTCGC AGTTAAGCAC 11881CCGTTGGGTA TACTTTGGCA TATGCGAGTC TCCCACCTAA TTGACGAAAT GGTCTCTCGC 11941CGCATTTACC GGACCATGGA ACATTCGGGT CAAGCGGCCT GGAAGCAGGT TGTTAGTGAA 12001GCCACTCTCA CAAAACTGTC AAGGCTTGAC GTAGTCACTC ATTTCCAACA CCTGGCCGCA 12061GTGGAGGCTG ATTCTTGCCG CTTCCTTAGC TCACGACTCG CGATGCTGAA AAACCTTGCC 12121GTTGGCAATG TGAGCCTGGA GTACAACACT ACTTTGGACC GCGTTGAGCT CATCTTTCCC 12181ACACCAGGTA CGAGGCCCAA GTTGACCGAT TTTAGGCAAT GGCTTATCAG CGTGCACGCT 12241TCCATCTTCT CCTCTGTGGC TTCGTCTGTT ACCTTGTTCA CAGTGCTTTG GCTTCGAATT 12301CCAGCTCTAC GCTATGTTTT TGGTTTCCAT TGGCCCACGG CAACACATCA TTCGAACTAASEQ ID NO: 22 Nucleotide encoding attenuated PRRSV 94881 ORF3 12219AT GGCTTATCAG CGTGCACGCT 12241TCCATCTTCT CCTCTGTGGC TTCGTCTGTT ACCTTGTTCA CAGTGCTTTG GCTTCGAATT 12301CCAGCTCTAC GCTATGTTTT TGGTTTCCAT TGGCCCACGG CAACACATCA TTCGAACTAA 12361CTATCAATTA CACTATATGT AAGCCATGCC CTACCAGTCA AGCTGCCCAA CAAAGACTCG 12421AGCCTGGCCG TAACGTGTGG TGCAAAATAG GGCACGACAG GTGTGAGGAA CGTGACCATG 12481ATGAGTTGTC AATGTCCATT CCGTCCGGGT ACGACAACCT CAAACTTGAG GGTTATTATG 12541CTTGGCTGGC TTTTTTGTCC TTTTCCTACG CGGCCCAATT CCATCCGGAG CTGTTCGGAA 12601TAGGAAACGT GTCGCGCGTC TTTGTGGATA AGCGACACCA GTTCATTTGC GCCGAGCATG 12661ATGGACAAAA TTCAACCATA TCTGCCAGAC ACAACATCTC CGCGTCGTAT GCGGTGTATT 12721ACCATCATCA AATAGACGGG GGCAATTGGT TTCATTTGGA ATGGCTGCGA CCATTCTTTT 12781CCTCCTGGCT GGTGCTCAAC ATCTCATGGT TTCTGAGGCG TTCGCCTGCA AGCCCTGCTT 12841CTCGACGCAT CTATCAGATA TTAAGACCAA CACGACCGCG GCTGCCGGTT TCATGGTCCT 12901TCAGAACATC AATTGTTTCC AATCTCACAG GGCCTCAACA GCGCAAGGTA CCACTCCCCT 12961CAGGAGGTCG TCCCAATGTC GTGAAGCCGT CGGCATTCCC CAGTACATCA CGATAASEQ ID NO: 23 Nucleotide encoding attenuated PRRSV 94881 ORF4 12761ATGGCTGCGA CCATTCTTTT 12781CCTCCTGGCT GGTGCTCAAC ATCTCATGGT TTCTGAGGCG TTCGCCTGCA AGCCCTGCTT 12841CTCGACGCAT CTATCAGATA TTAAGACCAA CACGACCGCG GCTGCCGGTT TCATGGTCCT 12901TCAGAACATC AATTGTTTCC AATCTCACAG GGCCTCAACA GCGCAAGGTA CCACTCCCCT 12961CAGGAGGTCG TCCCAATGTC GTGAAGCCGT CGGCATTCCC CAGTACATCA CGATAACGGC 13021TAATGTGACC GATGAATCGT ATTTGTACAA CGCGGACTTG CTGATGCTTT CCGCGTGCCT 13081TTTCTACGCC TCGGAAATGA GCGAGAAAGG CTTCAAAGTC ATCTTTGGGA ATATTTCTGG 13141CGTTGTTTCC GCTTGTGTTA ATTTCACAGA TTATGTGGCC CATGTGACCC AACACACTCA 13201GCAGCACCAT TTGGTAATTG ATCACATTCG GTTACTACAC TTCTTGACAC CGTCTACGAT 13261GAGGTGGGCT ACAACCATTG CTTGTTTGCT TGCCATTCTT TTGGCGGTAT GASEQ ID NO: 24 Nucleotide encoding attenuated PRRSV 94881 ORF5 13309AT GAAATGTTCT 13321TGCAAGTTGG GGCATTTCTT GACTCCTCAC TCTTGCTTCT GGTGGCTTTT TTTGCTGTGT 13381ACCGGCTTGT CTTGGTCCTT TGTCGATGGC AACGACGACA GCTCGACATC CCAATACATA 13441TATAATTTGA CGATATGCGA GCTGAATGGG ACCGAATGGT TGTCCGGTCA TTTTGATTGG 13501GCAGTCGAAA CCTTTGTGCT TTACCCAGTT GCCACTCATA TCATTTCACT GGGTTTTCTC 13561ACAACAAGCC ATTTCCTTGA TGCGCTCGGT CTCGGCGCTG TGTCCGCCAC AGGATTCATT 13621GGCGAGCGGT ATGTACTTAG CAGCATGTAC GGCGTTTGCG CCTTCGCGGC GTTCGTATGT 13681TTTGTCATCC GTGCTGCTAA AAATTGCATG GCTTGCCGCT ATGCCCGCAC CCGGTTTACC 13741AACTTCATCG TGGACGACCG GGGAAGAATC CATCGATGGA AGTCTTCAAT AGTGGTGGAG 13801AAATTGGGCA AAGCTGAAGT CGGTGGTGAC CTTGTCAACA TTAAGCATGT TGTCCTCGAA 13861GGGGTTAAAG CTCAACCTTT GACGAGGACT TCGGCTGAGC AATGGGAAGC CTAGSEQ ID NO: 25 Nucleotide encoding attenuated PRRSV 94881 ORF6 13902 ATGGGAAGC CTAGACGACT 13921TTTGCAACGA TCCCACCGCC GCACAAAAAC TCGTGCTGGC CTTTAGCATC ACATATACAC 13981CCATAATGAT ATACGCCCTT AAGGTGTCAC GCGGCCGACT CCTGGGGCTG TTGCACATCT 14041TGATATTTCT GAATTGTTCC TTTACTTTTG GGTACATGAC ATATGTGCAT TTTCAATCCA 14101CCAACCGTGT CGCATTCACT CTGGGGGCTG TAGTCGCCCT TTTGTGGGGT GTTTACAGCC 14161TCACAGAGTC ATGGAAGTTC ATCACTTCCA GATGCAGATT GTGTTGCCTA GGCCGGCGAT 14221ACATTCTGGC CCCTGCCCAT CACGTAGAAA GTGCTGCAGG CCTCCATTCA ATCCCAGCGT 14281CTGGTAACCG AGCATACGCT GTGAGAAAGC CCGGACTAAC ATCAGTGAAC GGCACTCTAG 14341TACCTGGGCT TCGGAGCCTC GTGCTGGGCG GCAAACGAGC TGTTAAACGA GGAGTGGTTA 14401ACCTCGTCAA GTATGGCCGG TAASEQ ID NO: 26 Nucleotide encoding attenuated PRRSV 94881 ORF7 14413  ATGGCCGG TAAGAACCAG AGCCAGAAGA AAAGAAGAAA TGCAGCTCCG 14461ATGGGGAAAG GCCAGCCAGT CAATCAACTG TGCCAGTTGC TGGGTACAAT GATAAAGTCC 14521CAGCGCCAGC AATCTAGGGG AGGACAGGCC AAAAAGAAGA AGCCTGAGAA GCCACATTTT 14581CCCCTAGCTG CTGAAGATGA CATTCGGCAC CATCTCACCC AGGCCGAACG TTCCCTCTGC 14641TTGCAATCGA TCCAGACGGC TTTCAATCAA GGCGCAGGAA CTGCGTCGCT TTCATCCAGC 14701GGGAAGGTCA GTTTCCAGGT TGAGTTCATG CTGCCGGTTG CTCATACAGT GCGCCTGATT 14761CGCGTGACTT CTACATCCGC CAGTCAGGGT GCAAATTAAT TTGACAGTCA GGTGAATGGC 14821CGCGATTGAC GTGTGGCCTC TAASEQ ID NO: 27 Nucleotide encoding parental PRRSV 94881 ORF1a 178 ATG 181TCTGGGATGT TCTCCCGGTG CATGTGCACC CCGGCTGCCC GGGTATTTTG GAACGCCGGC 241CAAGTCTATT GCACACGGTG TCTCAGTGCA CGGTCTCTTC TCTCTCCAGA ACTTCAGGAC 301ACGGACCTCG GTGCAGTTGG CTTGTTTCAC AAGCCTAAAG ACAAGCTCCA TTGGAAAGTT 361CCCATTGGTA TCCCCCAGGT GGAATGTTCT CCATCTGGGT GTTGCTGGCT GTCAACCATT 421TTTCCTTTAG CGCGCATGAC CTCCGGCAAT CACAACTTCC TTCAACGACT CGTGAAGGTT 481GCTGACGTAT TGTACCGTGA CGGTTGCTTA ACCCCTAGAC ACCTCCGTGA ACTCCAAGTT 541TACGAGCGTG GTTGCAATTG GTATCCGATT ACGGGGCCTG TGCCTGGGAT GGCTGTGTAC 601GCGAACTCCA TGCACGTGTC CGACCAACCG TTCCCTGGTG CCACTCATGT GTTAACAAAT 661TCCCCTTTGC CTCAACGGGC TTGTCGGCAG CCGTTCTGTC CGTTCGAAGA GGCCCATTCT 721AGCATATACA GGTGGGAAAA ATTTGTAATT TTTATGGATT CCTCCTCCGA CGGTCGATCT 781CGCATGATGT GGACTCCGGA ATCCGATGAC TCCACGGCTT TGGAAGTTCT GCCGCCCGAG 841CTAGAACACC AGGTCAAGGT CCTTGTTCGG AGCTTTCCCG CCCATCACCT TGTCGACCTT 901GCCGATTGGG AGCTCACTGA GTCCCCTGAG AACGGTTTTT CCTTCAGCAC GTCACATCCT 961TGCGGCTACC TTGTTCGGGA CCCGGCTGTA TCCGAAGGCA AGTGTTGGCT TTCCTGCTTT 1021TTGAGCCAGT CAGCCGAAGT GCTCAGTCGC GAGGCGCATC TGGCTACCGC CTATGGTTAC 1081CAAACCAAGT GGGGTGTGCC TGGCAAGTAC ATCCAGCGCA GACTTCAAGT TCACGGTCTC 1141CGTGCTGTGG TCGACCCTGA TGGTCCCATT CACGTTGAAG CATTGTCTTG CCCCCAGTCT 1201TGGATCAGGC ACTTGACCCT GAATGATGAT GTCACCCCGG GATTCGTTCG CCTAATGTCT 1261CTTCGCATTG TGCCGAACAC AGAGCCTACC ACACACCGGA TCTTTCGTTT TGGAGTGCAC 1321AAGTGGTATG GTGCCGCCGG CAAACGGGCC CGTGGCAAGC GTGCCGCCAA AAGTGAGAAA 1381GACTCGGCTT CCACCCTCAA GGTTGCCCGA CCGACTTCCA CCAGTGGAAT CGTCACCTAC 1441TCCCCACCTG CGGACGGGTC TTGTGGTTGG CATGCCCTTG CCGCCATACT GAACCGGATG 1501ATTAATAATG ACTTCACGTC CCCTCTGCCT CGGTACAACA GGCCGGAGGA CGATTGGGCT 1561TCTGATGGTG ACCTTGCTCA GGCCATTCAA TGTTTGCAAC TACCTGCCGC CATAGCTCGG 1621AACCGCGCCT GCCCTAACGC CAAATACCTC GTAAAACTCA ACGGAGTTCA TTGGGAGGTA 1681GAGGTGAGGC CTGGAATGGC TCCTCGCTCC CTCTCTCGTG AGTGCGTTGT TGGCGTCTGC 1741TCTGAAGGCT GTGTCGCGTC GCCTTACCCG GAGGACGGGT TGCCTAAACG TGCACTTGAG 1801GCCCTGGCGT CTGCTTATAG ACTGCCTTCA GACTGTGTTT GTGATGGTAT TATTGACTTC 1861CTTGCCAATC CACCTCCCCA GGAGTTCTGG ACTCTTGACA AAATGTTGAC TTCCCCGTCA 1921CCGGAGCAGT CCGGCTTCTC TAGTCTGTAT AAATTGTTGT TAGAGGTCTT GCCGCAGAAA 1981TGCGGATCCA CAGAAGGGGA ATTCATCTAT ACTGTTGAGA GGATGTTGAA GGATTGTCCG 2041AGCTCCAAAC AGGCCATGGC CCTCCTTGCA AAAATTAAGG TCCCATCCTC AAAGGCCCCA 2101TCCGTGACTC TGAACGAGTG CTTCCCCACG GATGTTCCAG TCAACTCTGA GTTAATATCT 2161TGGGAAGAGC CCAAAGACCC TGGCGCTGCT GTTGTCCTAT GTCCATCGGA TGCAAAAGAA 2221TCTAAGGAAA CAGCCCCTGA AGAAGCTCAA GCGAGAAACC GTAAGGTCCT CCACCCTGTG 2281GTCCTTACCG AGGAACTTAG CGAGCAACAG GTGCAGGTGG TTGAGGGTGA TCAGGATATG 2341CCACTGGATT TGACTTGGCC AACCTTAACC GCTACGGCGA CCCCTGTTAG AGGGCCGGTA 2401CCGGACAATT TGAGCTCTGG CATTGGTGCC CAGCCCGCTA CCGTTCAAGA ACTCATTCTG 2461GCGAGGCCTG CACCCCGTCT TGTTGAGCGC TGTGGCACGG AGTCGAACGG CAGCAGTTCA 2521TTTCTGGATT TGCCTGACGT GCAGACCTCG GACCAGCCTT TAGACCTGTC CCTGGCCGCG 2581TGGCCTGTAA GGGCTACCGC GTCTGACCCC GGTTGGATCC ACGGTAGGCG TGAGCCTGTC 2641TTTGTGAAGC CTCGAGGTGT TTTCTCTGAT GGCGAGTCGG CCCTTCAGTT CGGAGAGCTT 2701TCCGAAGCCA GTTCTGTCGT CGATGACCGG ACAAAAGAAG CTCCGGTGGT TGACGCCCCC 2761ATCGATTTGA CAACTTCGAA CGAGACGCTC TCTGGGTCTG ACCCCTTTGA ATTCGCCAAA 2821TTCAGGCGCC CGCGTTTCTC CGCGCAAGCT TTAATCGACC GAGGTGGTCC GCTTGCCGAT 2881GTTCATGCAA AGATAAAGAG TCGGGTATAT GAACAATGCC TTCAAGCTTG TGAACCTGGT 2941AGTCGTGCGA CCCCAGCCAC CAAGAAGTGG CTCGACAAAA TGTGGGACAG GGTGGACATG 3001AAAACTTGGC GCTGCACCTC GCAGTTCCAA GCTGGTCACA TTCTTGAGTC CCTCAAATTC 3061CTCCCTGACA TGATTCAAGA CACACCGCCT CCTGTTCCCA GGAAGAACCG AGCTGGTGAC 3121AGTGCCGGCC TGAAGCAACT GGTGGCGCAG TGGGATAGGA AATTGAGTGT GACACCCCCC 3181ACAAAACCGG TTGGACCGGT GCTTGACCAG ACCGTCCCTC TGCCTATGGA CATCCAGCAA 3241GAAGATGCCA TCTCCGCTGA CAAGCCACCC CATTCGCAAA ACCCTTCTAG TCAAGTAGAT 3301GTGGGTGGAG GTTGGAAAAG TTTTATGCTC TCCGGCACCC GTTTCGCGGG GTCCGTTAGT 3361CAGCGCCTTA CGACATGGGT TTTTGAGGTT CTCTCCCATC TCCCAGCTTT TATGCTCACA 3421CTTTTCTCGC CACGGGGCTC TATGGCTCCA GGTGATTGGC TGTTTGCAGG TGCTGTTCTA 3481CTTGCTCTCC TGCTCTGCCG TTCTTACCCA ATACTCGGAT GCCTTCCCTT ATTGGGTGTC 3541TTTTCTGGTT CTGTGCGGTG TGTTCGTTTG GGTGTTTTTG GTTCTTGGAT GGCTTTTGCT 3601GTATTTTTAT TCTCGACTCC ACCCGACCCA GTCGGTTCTT CTTGTGACCA CGATTCGCCG 3661GAGTGTCATG CTGAGCTTTT GGCTCTTGAG CAGCGCCAAC TTTGGGAACC TGTGCGCAGC 3721CTTGTGGTCG GGCCATCGGG CCTCTTATGC GTCATTCTTG GCAAGTTACT CGGTGGGTCA 3781CGTTGTCTCT GGTTTGTTCT CCTACGTATA TGCATGCTCG CAGATTTGGC AATTTCTCTT 3841ATTTATGTGG TGTCCCAAGG GCGTTGTCAC AAGTGTTGGG GAAAGTGTAT AAGGACGGCT 3901CCTGCAGAAG TGACCCTTAA TGTGTTTCCT TTTTCGCGCG CCACCCGCTC ATCTCTTGTG 3961TCCTTGTGTG ATCGGTTCCA AGCGCCAAAA GGAGTTGACC CCGTGCACTT GGCGACAGGC 4021TGGCGCGGGT GCTGGTGTGG TGAGAGCCCT ATTCATCAAT CACACCAAAA ACCGATAGCT 4081TATGCCAACT TGGATGAAAA GAAGATATCC GCCCAGACGG TGATTGCTGT CCCGTATGAT 4141CCCAGTCAGG CCATTAAATG CCTGAAAGTT TTGCAGGCAG GAGGGGCTAT TGTGGACCAG 4201CCTACGCCCG AGGTCGTCCG TGTGTCTGAG ATTCCCTTCT CGGCCCCATT TTTTCCGAAG 4261GTCCCAGTCA ACCCAGATTG CAGGGTTGTG GTAGATTCGG ACACTTTTGT GGCTGCGGTC 4321CGCTGCGGTT ATTCGACAGC ACAACTGGTC CTTGGTCGGG GCAACTTTGC CAAGCTAAAT 4381CAGACCCCCC TCAGGAACTC TGTCCCCACC AAAACAACTG GTGGGGCCTC ATACACCCTT 4441GCCGTGGCCC AGGTATCTGT GTGGACTCTT GTTCATTTCA TCCTCGGCCT TTGGTTAACG 4501TCACCTCAAG TGTGTGGTCG AGGGACCTCT GACCCGTGGT GTTCGAACCC TTTTTCGTAT 4561CCTACTTATG GCCCCGGAGT TGTGTGTTCC TCTCGACTCT GCGTGTCTGC CGACGGAGTT 4621ACCCTGCCAT TGTTCTCAGC CGTTGCCCAT CTTTCCGGTA GAGAGGTGGG GATTTTTATT 4681TTGGTGCTTG CCTCCTTGGG CGCTTTAGCC CACCGCTTGG CTCTTAAGGC AGACATGTCA 4741ATGGTCTTTT TGGCGTTTTG TGCTTACGCC TGGCCCATGA GCTCCTGGTT AATTTGCTTC 4801TTTCCTATGC TCTTGAGGTG GGTAACCCTT CATCCTCTCA CTATGCTTTG GGTGCACTCA 4861TTTTTGGTGT TTTGCCTACC AGCTGCCGGC GTTCTCTCGC TGGGAATAAC CGGTCTTCTT 4921TGGGCAGTTG GCCGTTTCAC CCAGGTTGCC GGAATTATCA CACCTTATGA CATCCACCAG 4981TATACCTCCG GACCACGTGG TGCAGCTGCT GTAGCAACGG CTCCAGAAGG TACTTACATG 5041GCGGCCGTTC GGAGAGCCGC TTTGACTGGA CGGACTTTGA TCTTCACACC ATCTGCAGTC 5101GGATCCCTTC TTGAAGGTGC TTTCAGAACT CAAAAGCCCT GCCTTAACAC CGTGAATGTC 5161GTAGGCTCTT CCCTTGGTTC TGGAGGAGTT TTCACCATTG ATGGCAGAAG AGTCATCGTC 5221ACTGCCACCC ATGTGTTGAA TGGTAACACA GCCAGGGTCA CTGGTGATTC CTACAACCGC 5281ATGCACACGT TCAATACTAA TGGTGATTAT GCCTGGTCCC ATGCTGATGA CTGGCAAGGC 5341GTTGCCCCTA TGGTTAAGAT CGCTAAGGGG TATCGCGGTC GTGCCTACTG GCAAACGTCA 5401ACCGGAGTCG AACCTGGCAT CATGGGGGAA GGATTCGCCT TCTGTTTCAC TAACTGTGGC 5461GACTCAGGGT CACCTGTCAT TTCAGAAGCT GGTGACCTTA TTGGAGTCCA TACCGGTTCA 5521AACAAACTCG GTTCTGGTCT TGTGACAACC CCTGAAGGGG AGACCTGCTC CATCAAGGAA 5581ACTAGGCTCT CTGACCTTTC TAGACATTTT GCAGGTCCAA GCGTCCCTCT TGGGGACATT 5641AAGTTGAGCC CAGCCATCAT CCCTGATGTG ACAACTATTC CGAGTGACTT GGCATCGCTC 5701CTTGCTTCTG TCCCCGTGAT GGAAGGTGGC CTCTCAACTG TCCAGCTTTT GTGCGTCTTT 5761TTCCTTCTCT GGCGCATGAT GGGCCATGCC TGGACACCCA TTGTTGCCGT AGGCTTCTTT 5821TTGCTGAATG AAATTCTCCC AGCAGTCTTG GTCCGAGCTG TGTTCTCTTT TGCACTCTTT 5881GTACTTGCAT GGGCCACCCC CTGGTCGGCA CAAGTGTTGA TGATTAGACT CCTCACGGCG 5941GCTCTCAACC GCAACAGGTT GTCCCTGGCG TTCTACGCAC TCGGAGGTGT CGTTGGCCTG 6001GCCACAGAAA TCGGGACTTT TGCTGGTGGA TGGCCTGAAC TGTCCCAAGC CCTCTCGACA 6061TACTGCTTCC TGCCCAGGTT CCTTGCTGTG ACTAGTTATG TCCCCACCAT CATCATCGGT 6121GGGCTCCATG CCCTCGGCGT AATTTTGTGG TTATTCAAAT ACCGATGCCT CCACAACATG 6181CTGGTTGGTG ATGGGAGTTT CTCAAGCGCT TTCTTCCTAC GGTATTTTGC TGAGGGTAAT 6241CTTAGGAAAG GCGTGTCGCA GTCCTGTGGC ATGAATAACG AATCCCTGAC AGCTGCTTTG 6301GCTTGCAAGT TGTCGCAAGC TGACCTTGAT TTTTTGTCCA GTTTAACGAA CTTCAAGTGC 6361TTTGTGTCCG CTTCAAACAT GAAAAATGCA GCTGGCCAAT ACATCGAGGC GGCGTATGCT 6421AGAGCTCTGC GTCAGGAGCT GGCCTCCTTG GTTCAGGTTG ACAAGATGAA AGGAGTATTG 6481GCCAAGCTCG AGGCTTTCGC TGAGACGGCC ACTCCGTCAC TTGACACAGG TGACGTGATT 6541GTTCTGCTTG GGCAACACCC CCATGGATCC ATCCTCGACA TTAATGTGGG GGGTGAAAGG 6601AAAACTGTGT CTGTGCAAGA AACACGATGC CTGGGTGGTT CCAAATTCAG TGTCTGCACT 6661GTCGTGTCCA ACACGCCCGT GGATACCTTG ACCGGCATCC CACTTCAGAC GCCAACCCCA 6721CTTTTTGAAA ATGGCCCGCG CCATCGCAGC GAGGACGACG ACCTTAAAGT TGAGAGAATG 6781AAAAAACACT GTGTATCCCT CGGCTTCCAC AAAATCAATG GTAAAGTTTA CTGCAAAATT 6841TGGGACAAGT CTAACGGCGA CACCTTTTAC ACGGATGATT CCCGATACAC TCAAGACCAT 6901GCTTTTCAGG ACAGGTCAAC CGACTATAGA GACAGGGATT ATGAAGGTGT ACAGACCGCC 6961CCCCAACAGG GATTCGATCC AAAGTCCGAA GCCCCTGTTG GCACTGTTGT AATCGGTGGC 7021ATTACGTATA ACAGGCATCT GGTCAAAGGT AAGGAGGTCC TAGTTCCCAA ACCTGACAAC 7081TGCCTTGAAG CTGCCAGACT GTCCCTTGAG CAAGCTCTTG CTGGGATGGG CCAAACTTGT 7141GACCTTACAG CTACCGAAGT GGAGAAACTA AAGCGCATCA TTAGTCAACT CCAAGGTCTG 7201ACCACTGAAC AGGCTTTAAA CTGCTAGCCG CCAGCGGCTT GACCCGCTGT GGCCGCGGCGSEQ ID NO: 28 Nucleotide encoding parental PRRSV 94881 ORF1b 7209        AC AGGCTTTAAA CTGCTAGCCG CCAGCGGCTT GACCCGCTGT GGCCGCGGCG 7261GCCTAGTTGT AACTGAAACG GCGGTAAAAA TCGTAAAATA CCACAGCAGA ACTTTCACCT 7321TAGGCTCTTT AGACCTAAAA GTCACCTCCG AGGTGGAGGT GAAGAAATCA ACTGAGCAGG 7381GGCACGCTGT CGTGGCGAAC TTATGTTCCG GTGTCGTCTT GATGAGGCCT CACCCACCGT 7441CCCTTGTTGA CGTTCTCCTC AAACCCGGAC TTGACACAAC ACCCGGCATT CAACCAGGGC 7501ATGGGGCCGG GAATATGGGC GTGAACGGTT CTATTTGGGA TTTTGAAACT GCACCCACAA 7561AGGTAGAACT AGAGTTGTCC AAGCAAATAA TCCAAGCATG TGAAGTCAGG CGCGGGGACG 7621CCCCTAACCT CCAACTCCCC TACAAGCTTT ATCCTGTCAG GGGGGACCCC GAGCGGCGTA 7681AAGGTCGCCT TGTCAACACT AGGTTTGGAG ATTTACCTTA CAAAACTCCC CAAGACACCA 7741AGTCCGCAAT TCATGCGGCT TGTTGCCTGC ATCCCAATGG GGTCCTCGTG TCTGATGGTA 7801AATCCACGCT GGGTACCACT CTTCAACATG GTTTCGAGCT TTATGTCCCC ACTGTACCTT 7861ATAGTGTCAT GGAATACCTT GATTCACGCC CTGACACCCC TTTTATGTGT ACTAAACATG 7921GCACTTCCAA GGCTGCTGCA GAGGACCTCC AAAAATATGA CCTATCCACT CAAGGGTTTG 7981TCTTGCCTGG GGTCCTACGC CTAGTGCGCA GGTTCATCTT TAGCCATGTT GGTAAGGCGC 8041CACCACTGTT CCTTCCATCA ACCTACCCTG CCAAGAACTC CATGGCAGGG GTCAATGGCC 8101AGAGGTTCCC AACAAAGGAT GTCCAGAGCA TACCTGAAAT TGATGAAATG TGCGCCCGTG 8161CCGTCAAGGA AAATTGGCAG ACTGTGACAC CTTGCACCCT CAAAAAACAG TACTGTTCCA 8221AACCTAAAAC TAGAACCATC CTAGGTACCA ACAACTTCAT AGCCTTGGCT CACAGGTCAG 8281CACTCAGTGG TGTCACCCAG GCGTTCATGA AGAAGGCCTG GAAGTCCCCA ATTGCCTTGG 8341GGAAAAACAA GTTTAAGGAA TTGCATTGCA CTGTCGCCGG CAGATGCCTT GAGGCTGACC 8401TGGCTTCCTG CGATCGCAGC ACCCCCGCCA TTGTGAGGTG GTTTGTTGCC AACCTCCTGT 8461ATGAACTTGC AGGATGTGAA GAGTACTTGC CTAGCTACGT GCTCAACTGT TGCCATGACC 8521TTGTGGCAAC GCAGGATGGC GCTTTCACAA AACGCGGTGG CCTGTCGTCC GGGGACCCCG 8581TCACCAGTGT GTCCAACACC GTCTACTCAC TGATAATTTA CGCCCAGCAC ATGGTGCTTT 8641CGGCCTTGAA GATGGGTCAT GAAATTGGTC TCAAGTTCCT TGAGGAACAG CTCAAATTTG 8701AGGACCTTCT TGAAATCCAG CCCATGTTAG TGTATTCTGA TGACCTCGTC TTGTATGCGG 8761AAAGACCCAC TTTTCCCAAC TACCATTGGT GGGTCGAGCA TCTTGACCTG ATGTTGGGCT 8821TTAAAACGGA CCCAAAGAAA ACTGTCATAA CTGATAAACC CAGTTTTCTC GGCTGCAGAA 8881TTGAAGCAGG ACGGCAGTTA GTCCCCAATC GCGACCGTAT TCTGGCTGCT CTTGCATATC 8941ATATGAAGGC GCAGAACGCC TCAGAGTATT ATGCGTCCGC TGCCGCAATT CTGATGGATT 9001CGTGTGCTTG CATTGACCAT GACCCCGAGT GGTATGAGGA CCTTATCTGC GGCATCGCCC 9061GGTGTGCTCG CCAGGACGGT TACCGTTTTC CAGGCCCGGC ATTTTTCATG TCCATGTGGG 9121AGAAGCTGAA AAGTCATAAC GAAGGGAAGA AATGCCGTCA CTGCGGCATC TGCGACGCCA 9181AAGCCGACTA TGCGTCCGCC TGTGGACTTG ATTTGTGTTT GTTCCATTCA CACTTTCATC 9241AACACTGCCC AGTCACTCTG AGCTGTGGCC ACCATGCCGG TTCAAAGGAA TGTTCGCAGT 9301GTCAGTCACC TGTCGGGGCT GGCAAATCCC CCCTTGACGC TGTGCTGAAA CAAATCCCGT 9361ACAAACCTCC TCGTACCATT ATCATGAAGG TGGACAACAA AACAACGACC CTTGACCCGG 9421GAAGATATCA GTCCCGTCGA GGTCTTGTTG CAGTCAAAAG AGGTATTGCA GGTAATGAGG 9481TTGATCTTTC TGATGGAGAC TACCAAGTGG TGCCTCTTTT GCCGACTTGC AAAGACATAA 9541ACATGGTGAA GGTGGCTTGC AACGTACTAC TCAGCAAGTT TATAGTAGGG CCGCCAGGTT 9601CCGGAAAAAC CACCTGGCTA CTGAACCAAG TCCAGGACGA TGATGTCATT TACACACCTA 9661CTCATCAGAC AATGTTTGAC ATAGTCAGTG CTCTTAAAGT TTGCAGGTAT TCCATCCCAG 9721GAGCCTCAGG ACTCCCTTTT CCACCACCTG CCAGGTCCGG GCCGTGGGTT AGGCTCATCG 9781CCAGCGGACA TGTCCCTGGC CGAGTGTCAT ATCTCGATGA GGCAGGATAT TGCAATCATC 9841TAGACATTCT AAGGCTGCTT TCCAAAACAC CCCTTGTGTG TTTGGGTGAC CTTCAGCAAC 9901TTCACCCGGT CGGCTTTGAT TCCTATTGTT ATGTGTTCGA TCAGATGCCT CAGAAGCAGC 9961TGACCACCAT TTATAGATTT GGCCCTAACA TCTGTGCAGC CATCCAGCCT TGTTACAGGG 10021AGAAACTTGA ATCCAAGGCC AGGAACACCA GAGTGGTTTT CACCACCCGG CCTGTGGCCT 10081TTGGTCAGGT CCTGACACCG TACCACAAAG ATCGTACCGG CTCTGCAATA ACTATAGATT 10141CATCCCAGGG GGCGACCTTC GACATTGTGA CATTGCATCT ACCATCGCCA AAGTCCCTAA 10201ACAAATCCCG AGCACTTGTA GCCATCACTC GGGCAAGACA TGGGTTGTTC ATTTATGACC 10261CTCATGACCA ACTCCAGGAG TTTTTCAACT TAACCCCCGA GCGCACTGAT TGTAACCTTG 10321CGTTCAGCCG TGGGGATGAG CTGGTTGTTT TGAATGTGGA TAATGCGGTC ACAACTGTAG 10381CGAAGGCCCT AGAGACAGGT TCACCCCGAT TTCGAGTATC GGACCCGAGG TGCAAGTCTC 10441TCTTAGCCGC TTGTTCGGCC AGTCTAGAAG GGAGCTGCAT GCCACTACCA CAAGTAGCAC 10501ATAACCTGGG GTTTTACTTT TCCCCGGACA GCCCAGCTTT TGCACCCCTG CCAAAAGAGC 10561TGGCGCCACA TTGGCCAGTG GTCACCCACC AGAATAATCG AGCGTGGCCT GATCGACTTG 10621TCGCTAGTAT GCGCCCAATT GATGCCCGCT ACAGCAAGCC AATGGTCGGT GCAGGGTATG 10681TGGTCGGGCC ATCCATTTTT CTTGGCACTC CTGGTGTGGT GTCATACTAT CTCACATTAT 10741ACATCGGGGG CGAGCCTCAG GCCCTGCCAG AAACACTCGT TTCAACAGGA CGTATAGCCA 10801CAGATTGTCG GGAATATCTC GACGCGGCTG AGGAAGAGGC AGCGAGAGAA CTTCCCCACG 10861CATTTATTGG CGATGTCAAA GGCACTACGG TCGGGGGGTG TCACCACATT ACATCGAAAT 10921ACCTACCTAG GTCCCTGCCT AAAGACTCTG TTGCTGTGGT TGGGGTGAGT TCGCCCGGTA 10981GGGCTGCTAA AGCCGTGTGC ACTCTCACCG ATGTGTACCT CCCCGAACTC CGACCATATT 11041TGCAACCGGA GACGGCATCA AAATGCTGGA AACTTAAACT GGATTTCAGG GATGTTCGAC 11101TGATGGTCTG GAAAGGCGCC ACAGCCTATT TCCAGTTGGA AGGGCTGACA TGGTCAGCGC 11161TGCCCGATTA TGCTAGGTTC ATTCAGCTAC CCAAGGATGC CGTTGTGTAC ATCGATCCGT 11221GTATAGGGCC GGCAACAGCC AATCGCAAGG TTGTGCGAAC CACAGACTGG CGGGCCGACC 11281TGGCAGTGAC ACCGTATGAT TACGGTGCTC AGGTCATTTT GACAACAGCC TGGTTCGAGG 11341ACCTTGGGCC GCAGTGGAAG ATTTTGGGGT TGCAGCCTTT CAGACGAACA TTTGGCTTTG 11401AGAACACTGA AGATTGGGCA ATTCTCGCAC GCCGTATGAA TGACGGCAAA GATTACACTG 11461ACTATAATTG GCATTGTGTA CGAGAACGCC CACACGCAAT TTACGGGCGC GCCCGTGACC 11521ATACGTATCA TTTTGCCCTT GGCACTGAAC TGCAAGTAGA GCTGGGCAGA CCCCGGCTGC 11581CTCCTGAGCA AGTGCCGTGASEQ ID NO: 29 Nucleotide encoding parental PRRSV 94881 ORF2 11611ATGCAATGGG TTCACTGTGG AGTAAAATCA 11641GTCAGTTGTT CGTGGATGCC TTCACTGAGT TCCTTGTTAG TGTGGTTGAC ATTGTCATCT 11701TTCTCGCCAT ATTGTTTGGG TTCACTGTTG CAGGCTGGTT ATTGGTCTTC CTTCTCAGAG 11761TGGTTTGCTC CGCGTTTCTC CGTTCGCGCT CTGCCATTCA CTCTCCCGAA CTATCGAAGG 11821TCCTATGAGG GCTTGCTACC CAACTGCAGA CCGGATGTCC CACAATTCGC AGTTAAGCAC 11881CCGTTGGGTA TACTTTGGCA TATGCGAGTC TCCCACCTAA TTGACGAAAT GGTCTCTCGC 11941CGCATTTACC GGACCATGGA ACATTCGGGT CAAGCGGCCT GGAAGCAGGT TGTTAGTGAA 12001GCCACTCTCA CAAAACTGTC AAGGCTTGAC GTAGTCACTC ATTTCCAACA CCTGGCCGCA 12061GTGGAGGCTG ATTCTTGCCG CTTCCTTAGC TCACGACTCG CGATGCTGAA AAACCTTGCC 12121GTTGGCAATG TGAGCCTGGA GTACAACACT ACTTTGGACC GCGTTGAGCT CATCTTTCCC 12181ACACCAGGTA CGAGGCCCAA GTTGACCGAT TTTAGGCAAT GGCTTATCAG CGTGCACGCT 12241TCCATCTTCT CCTCTGTGGC TTCGTCTGTT ACCTTGTTCA CAGTGCTTTG GCTTCGAATT 12301CCAGCTCTAC GCTATGTTTT TGGTTTCCAT TGGCCCACGG CAACACATCA TTCGAACTAASEQ ID NO: 30 Nucleotide encoding parental PRRSV 94881 ORF3 12219        AT GGCTTATCAG CGTGCACGCT 12241TCCATCTTCT CCTCTGTGGC TTCGTCTGTT ACCTTGTTCA CAGTGCTTTG GCTTCGAATT 12301CCAGCTCTAC GCTATGTTTT TGGTTTCCAT TGGCCCACGG CAACACATCA TTCGAACTAA 12361CTATCAATTA CACTATATGT AAGCCATGCC CTACCAGTCA AGCTGCCCAA CAAAGACTCG 12421AGCCTGGCCG TAACGTGTGG TGCAAAATAG GGCACGACAG GTGTGAGGAA CGTGACCATG 12481ATGAGTTGTC AATGTCCATT CCGTCCGGGT ACGACAACCT CAAACTTGAG GGTTATTATG 12541CTTGGCTGGC TTTTTTGTCC TTTTCCTACG CGGCCCAATT CCATCCGGAG CTGTTCGGAA 12601TAGGAAACGT GTCGCGCGTC TTTGTGGATA AGCGACACCA GTTCATTTGC GCCGAGCATG 12661ATGGACAAAA TTCAACCATA TCTGCCAGAC ACAACATCTC CGCGTCGTAT GCGGTGTATT 12721ACCATCATCA AATAGACGGG GGCAATTGGT TTCATTTGGA ATGGCTGCGA CCATTCTTTT 12781CCTCCTGGCT GGTGCTCAAC ATCTCATGGT TTCTGAGGCG TTCGCCTGCA AGCCCTGCTT 12841CTCGACGCAT CTATCAGATA TTAAGACCAA CACGACCGCG GCTGCCGGTT TCATGGTCCT 12901TCAGAACATC AATTGTTTCC AATCTCACAG GGCCTCAACA GCGCAAGGTA CCACTCCCCT 12961CAGGAGGTCG TCCCAATGTC GTGAAGCCGT CGGCATTCCC CAGTACATCA CGATAASEQ ID NO: 31 Nucleotide encoding parental PRRSV 94881 ORF4 12761ATGGCTGCGA CCATTCTTTT 12781CCTCCTGGCT GGTGCTCAAC ATCTCATGGT TTCTGAGGCG TTCGCCTGCA AGCCCTGCTT 12841CTCGACGCAT CTATCAGATA TTAAGACCAA CACGACCGCG GCTGCCGGTT TCATGGTCCT 12901TCAGAACATC AATTGTTTCC AATCTCACAG GGCCTCAACA GCGCAAGGTA CCACTCCCCT 12961CAGGAGGTCG TCCCAATGTC GTGAAGCCGT CGGCATTCCC CAGTACATCA CGATAACGGC 13021TAATGTGACC GATGAATCGT ATTTGTACAA CGCGGACTTG CTGATGCTTT CCGCGTGCCT 13081TTTCTACGCC TCGGAAATGA GCGAGAAAGG CTTCAAAGTC ATCTTTGGGA ATATTTCTGG 13141CGTTGTTTCC GCTTGTGTTA ATTTCACAGA TTATGTGGCC CATGTGACCC AACACACTCA 13201GCAGCACCAT TTGGTAATTG ATCACATTCG GTTACTACAC TTCTTGACAC CGTCTACGAT 13261GAGGTGGGCT ACAACCATTG CTTGTTTGTT TGCCATTCTT TTGGCGGTAT GASEQ ID NO: 32 Nucleotide encoding parental PRRSV 94881 ORF5 13309AT GAAATGTTCT 13321TGCAAGTTGG GGCATTTCTT GACTCCTCAC TCTTGCTTCT GGTGGCTTTT TTTGCTGTGT 13381ACCGGCTTGT CTTGGTCCTT TGTCGATGGC AACGACAACA GCTCGACATC CCAATACATA 13441TATAATTTGA CGATATGCGA GCTGAATGGG ACCGAATGGT TGTCCGGTCA TTTTGATTGG 13501GCAGTCGAAA CCTTTGTGCT TTACCCAGTT GCCACTCATA TCATTTCACT GGGTTTTCTC 13561ACAACAAGCC ATTTCCTTGA TGCGCTCGGT CTCGGCGCTG TGTCCGCCAC AGGATTCATT 13621GGCGAGCGGT ATGTACTTAG CAGCATGTAC GGCGTTTGCG CCTTCGCGGC GCTCGTATGT 13681TTTGTCATCC GTGCTGCTAA AAATTGCATG GCTTGCCGCT ATGCCCGCAC CCGGTTTACC 13741AACTTCATCG TGGACGACCG GGGAAGAATC CATCGATGGA AGTCTTCAAT AGTGGTGGAG 13801AAATTGGGCA AAGCTGAAGT CGGTGGTGAC CTTGTCAACA TTAAGCATGT TGTCCTCGAA 13861GGGGTTAAAG CTCAACCCTT GACGAGGACT TCGGCTGAGC AATGGGAAGC CTAGSEQ ID NO: 33 Nucleotide encoding parental PRRSV 94881 ORF6 13902 ATGGGAAGC CTAGACGACT 13921TTTGCAACGA TCCCACCGCC GCACAAAAAC TCGTGCTGGC CTTTAGCATC ACATATACAC 13981CCATAATGAT ATACGCCCTT AAGGTGTCAC GCGGCCGACT CCTGGGGCTG TTGCACATCT 14041TGATATTTCT GAATTGTTCC TTTACTTTTG GGTACATGAC ATATGTGCAT TTTCAATCCA 14101CCAACCGTGT CGCACTCACT CTGGGGGCTG TAGTCGCCCT TTTGTGGGGT GTTTACAGCC 14161TCACAGAGTC ATGGAAGTTC ATCACTTCCA GATGCAGATT GTGTTGCCTA GGCCGGCGAT 14221ACATTCTGGC CCCTGCCCAT CACGTAGAAA GTGCTGCAGG CCTCCATTCA ATCCCAGCGT 14281CTGGTAACCG AGCATACGCT GTGAGAAAGC CCGGACTAAC ATCAGTGAAC GGCACTCTAG 14341TACCTGGGCT TCGGAGCCTC GTGCTGGGCG GCAAACGAGC TGTTAAACGA GGAGTGGTTA 14401ACCTCGTCAAGTATGGCCGGT AASEQ ID NO: 34 Nucleotide encoding parental PRRSV 94881 ORF7 14413  ATGGCCGG TAAGAACCAG AGCCAGAAGA AAAGAAGAAA TGCAGCTCCG 14461ATGGGGAAAG GCCAGCCAGT CAATCAACTG TGCCAGTTGC TGGGTACAAT GATAAAGTCC 14521CAGCGCCAGC AATCTAGGGG AGGACAGGCC AAAAAGAAGA AGCCTGAGAA GCCACATTTT 14581CCCCTAGCTG CTGAAGATGA CATTCGGCAC CATCTCACCC AGGCCGAACG TTCCCTCTGC 14641TTGCAATCGA TCCAGACGGC TTTCAATCAA GGCGCAGGAA CTGCGTCGCT TTCATCCAGC 14701GGGAAGGTCA GTTTCCAGGT TGAGTTCATG CTGCCGGTTG CTCATACAGT GCGCCTGATT 14761CGCGTGACTT CTACATCCGC CAGTCAGGGT GCAAATTAA

1. A method of immunizing swine against porcine reproductive andrespiratory syndrome (PRRS), said method comprising the step ofadministering to swine a vaccine composition including a live porcinereproductive and respiratory syndrome virus mixed with apharmacologically compatible carrier agent, said virus comprising PRRS94881 virus passaged at least 36 times in cell culture to modify thevirus such that when the modified virus is administered to a swine orother mammal prone to PRRS, it fails to cause clinical signs of PRRSdisease but is capable of inducing an immune response that immunizes themammal against pathogenic forms of PRRS.
 2. The method of claim 1,wherein said swine presents no lung lesions after vaccination.
 3. Themethod of claim 1, wherein said swine presents fewer lung lesions aftervaccination as compared to vaccination with Porcilis vaccine.
 4. A PRRSvirus having a nucleotide sequence that is at least 95% homologous withthe sequence set forth in either SEQ ID NO:1 or SEQ ID NO:10.
 5. A PRRSvirus that comprises at least one ORF that encodes a protein that is atleast 98% identical to any of the sequences set forth in SEQ ID NO: 2 to9 or SEQ ID NO:11 to SEQ ID NO:18.
 6. A PRRS virus that has a nucleotidesequence of SEQ ID NO:1 or SEQ ID NO:10 or a fragment of either SEQ IDNO:1 or SEQ ID NO:2 wherein said fragment encodes an ORF selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, and SEQ ID NO:18.
 7. A subunit vaccine for vaccination ofa porcine animal wherein said vaccine comprises one or more nucleotidesselected from the group consisting of a nucleotide that encodes an ORFselected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15,SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.
 8. A subunit vaccine forvaccination of a porcine animal wherein said vaccine comprises one ormore nucleotides selected from the group consisting of SEQ ID NO:19; SEQID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ ID NO:24; SEQ IDNO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ ID NO:29; SEQ IDNO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; and SEQ ID NO:34.
 9. Acomposition comprising one or more proteins selected from the groupconsisting of a protein having the sequence of SEQ ID NO:2, SEQ ID NO:3,SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ IDNO:15, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID NO:18.
 10. An isolatednucleic acid comprising a sequence selected from the group consisting ofSEQ ID NO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23;SEQ ID NO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28;SEQ ID NO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33;and SEQ ID NO:34.
 11. A recombinant expression vector comprising anucleic acid sequence that encodes one or more PRRSV ORFs selected fromthe group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, and SEQ ID NO:18 operably linked to a promoter.
 12. Therecombinant expression vector of claim 11 wherein said nucleic acidencoding said ORFs is selected from the group consisting of SEQ IDNO:19; SEQ ID NO:20; SEQ ID NO:21; SEQ ID NO:22; SEQ ID NO:23; SEQ IDNO:24; SEQ ID NO:25; SEQ ID NO:26; SEQ ID NO:27; SEQ ID NO:28; SEQ IDNO:29; SEQ ID NO:30; SEQ ID NO:31; SEQ ID NO:32; SEQ ID NO:33; and SEQID NO:34.
 13. A vaccine comprising a recombinant expression vector ofclaim
 11. 14. A vaccine comprising a recombinant expression vector ofclaim 12.